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CONFIDENTIAL TREATMENT REQUESTED
by LXR Biotechnology Inc.
Located at 0000 Xxxxxx Xxx Xxxxx
Xxxxxxxx, XX 00000
Exhibit 10.33
LICENSE AGREEMENT
LICENSE AGREEMENT dated as of August 29, 1996, between, LXR
BIOTECHNOLOGY INC., a Delaware corporation, on behalf of itself and OPTICAL
ANALYTIC, INC., its wholly-owned subsidiary ("Licensor"), and THE XXXXXX-XXXXX
CORPORATION, a New York corporation ("Licensee").
W I T N E S S E T H :
WHEREAS, Licensor has developed and patented or filed patent
applications or otherwise obtained the rights to certain technology related to
laser scanning digital imaging; and
WHEREAS, Licensor has developed or otherwise obtained the rights to
certain software related to laser scanning digital imaging; and
WHEREAS, Licensor has skill and knowledge ("know-how") in the practice
of such technology; and WHEREAS, Licensee desires to obtain a license from
Licensor to use such patented technology, software and know-how, and Licensor is
willing to grant such a license, subject to the terms and conditions of this
Agreement.
NOW, THEREFORE, in consideration of the foregoing and of the mutual
covenants hereinafter set forth, the parties hereto agree as follows:
ARTICLE I
DEFINITIONS
Unless the context otherwise requires, the following terms, for all
purposes of this Agreement, shall have the meanings specified in this Section
1.1.
1.1 "Affiliate" shall mean a Person who, directly or indirectly,
controls, is controlled by, or is under common control with the specified
entity.
1.2 "Agreement" shall mean this Agreement, all Schedules annexed
hereto, and all properly executed amendments hereto.
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1.3 "Confidential Information" shall include verbal information and
documentation in writing concerning the practice and use of or embodying the
Intellectual Property and Improvements thereto. Such documentation shall
include, without limitation any technical drawings, designs, computer programs,
algorithms, formulas, diagrams, flow charts, application information,
specifications, notebooks, tracings, photographs, reports, findings,
recommendations, manuals or other materials concerning the design, development,
manufacture and/or use of the Licensed Patents, Licensed Patent Applications,
Proprietary Information and Licensed software in any medium.
1.4 "Effective Date" shall mean August 29, 1996.
1.5 "Field of Use" shall mean any area of business activity in which
the Intellectual Property can be or is being developed and/or commercialized
related to hybridization based assays for life science research and any other
life science or clinical diagnostic application. Field of Use is subject to
Section 9.6 herein.
1.6 "Final Adjudication" shall mean an adjudication, by a trial court
or court of appeal of competent jurisdiction, which adjudication shall be final,
binding and unappealable, whether by its terms or the passage of time.
1.7 "Improvement(s)" shall mean any modifications, improvements, or
developments, whether or not patentable, copyrightable or protectable under any
trade secret law or any other domestic or foreign statutory or legal principle
now or hereafter defining, creating or protecting any interest in intellectual
property, solely within the Field of Use, which (i) improve the performance of
any of the inventions described in the Licensed Patents; (ii) modify a component
(or material) useful in the inventions described in the Licensed Patents; (iii)
add functions to the Licensed Patents; or (iv) improve, update, or enhance the
software and that are developed by Licensor and made generally available by
Licensor without a separate charge to licensees of the Licensed Products.
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1.8 "Infringing" and "Infringement" shall mean any infringement of any
patent, any copyright, any misappropriation of any trade secrets or know-how, or
any other violation of any intellectual property right of any Person.
1.9 "Infringer" shall mean a Person who Infringes.
1.10 "Intellectual Property" shall mean the Licensed Patents, Licensed
Patent Applications, Licensed Software and the Proprietary Information,
collectively.
1.11 "Liabilities" shall mean all liabilities, damages, losses, costs
and expenses (including court costs and reasonable attorneys' fees and experts'
fees.)
1.12 "Licensed Patents" shall mean the Patents set forth in Schedule
1.12 hereto, individually or collectively, and all divisions, reissues,
continuations, continuations-in-part and extensions thereof and corresponding
foreign patents and patent applications. Copies of the Licensed Patents are
included in Exhibit C hereto.
1.13 "Licensed Patent Applications" shall mean the United States patent
applications set forth in Schedule 1.13 hereto, individually or collectively,
and all divisions, reissues, continuations, continuations-in-part and extensions
thereof and corresponding foreign patents and patent applications. Any patents
which issue from the patent applications set forth in Schedule 1.13 shall be
deemed to be Licensed Patents as of the date of such issue. Copies of the
Licensed Patent Applications are included in Exhibit C hereto.
1.14 "Licensed Products" shall mean any and all products containing a
light generating component, a light collecting component, a light directing
component or a light detecting component the functional combination of which is
covered by one or more Subsisting Claims, together with the underlying
electronics and firmware necessary for the coordination and control of the
foregoing components and/or Licensed Software (object code form).
1.15 "Licensed Software" shall mean the copyrighted software programs
as set forth in Schedule 1.15 for use with Licensed Products in object code form
within the Field of Use. Except for purposes of Article II hereof, the term
`Licensed Software' shall also mean the Source Code for such object code (the
"Licensed Source Code").
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1.16 "Net Sales" shall mean (i) the gross proceeds received by Licensee
and sublicensees thereof from the sale or lease of the Licensed Products minus
(A) all returns of Licensed Products, (B) customary discounts made available by
Licensee to customers for the Licensed Products, and (C) all charges incurred by
Licensee for the shipping and handling of the Licensed Products. Gross proceeds
shall not be diminished by any present or future taxes and levies, imposts,
fees, assessments, deductions, or charges and withholdings whatsoever imposed,
assessed, levied or collected by any political subdivision or taxing authority.
1.17 "Ohio State License Agreement" shall mean the License Agreement
dated August 15, between, Optical Analytic, Inc., The Ohio State University and
The Ohio State University Research Foundation as amended May 21, 1993, September
7, 1993 November 29, 1993, July 10, 1996 and August __, 1996 (not executed)
[copies attached hereto as Exhibit B] or at any other time, relating to the
license of the Licensed Patents.
1.18 "Person" shall mean any natural person, corporation, partnership,
trust, joint venture or other entity.
1.19 "Proprietary Information" shall mean the Licensor's trade secrets
and know-how relating to the Licensed Patents, Licensed Patent Applications and
Licensed Software. Proprietary Information includes without limitation the
listing of categories involving transfer of technology and trade secrets as set
forth in Schedule 1.19.
1.20 "Source Code" shall mean a series of instructions or statements in
a computer programming language which is normally readily read by humans trained
in that language, and which is normally transformed by an interpreter or
compiler into object code for actual use on a computer.
1.21 "Subsisting Claim" shall mean any claim under a Licensed Patent
required to practice the invention covered thereby, which claim has not expired
or lapsed, or been abandoned, canceled, disclaimed, awarded to another party in
an interference proceeding, or declared invalid by a court of competent
jurisdiction in a Final Adjudication.
1.22 "Term" shall mean the term of this Agreement pursuant to Section
9.1 hereof.
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ARTICLE II
LICENSE RIGHTS
2.1 License Grant. Subject to the terms and conditions of this
Agreement, Licensor hereby grants to Licensee for the Term and, within the Field
of Use, an exclusive, worldwide license under the Licensed Patents and Licensed
Patent Applications to make, have made, use, sell or lease the Licensed
Products. Licensor further grants to Licensee for the Term and, within the Field
of Use, an exclusive worldwide license for the use of the Licensed Software in
object code form and Proprietary Information to make, have made, use, sell or
lease the Licensed Products. Licensor does not grant to Licensee an independent
license to sell or distribute the Licensed Software apart from the executable
form incorporated in or otherwise as a unit within the Licensed Products.
2.2 Sublicensing. Licensee may sublicense to any Person all or a
portion of its rights hereunder subject to review by Licensor with respect to
sublicenses for manufacturing of the Licensed Products. Licensor shall not
unreasonably withhold the right to sublicense. Sublicense of the Licensed
Software is limited to executable form accompanying the sale of the Licensed
Products and Sublicensee agrees not to distribute the Licensed Product
containing an object code version of the Licensed Software other than pursuant
to a valid license agreement containing substantially the term that, "End User
will not adapt, translate, reverse engineer, decompile, disassemble or create
derivative works based on the Licensed Software or any part thereof".
2.3 Source Code License Grant.
(a) Subject to the terms and conditions of this Agreement,
Licensor hereby grants to Licensee for the term and within the Field of Use, a
non-exclusive, worldwide license to modify the Licensed Source Code for internal
purposes and only to the extent reasonably necessary for Licensee to exercise
fully the object code license granted in Section 2.1 hereof, including without
limitation the making of Improvements. Licensee shall have the right to make and
retain copies of such Licensed Source Code only to the extent necessary for the
exercise of
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the license granted herein, and shall destroy all copies that become
unnecessary. Licensor does not grant to Licensee any license to use or modify
the Licensed Source Code outside of the scope of the object code license, or to
sell or distribute the Licensed Source Code, or to sublicense to any third party
the license granted to Licensee in this Section 2.3. Licensor does not grant to
Licensee an independent license to sell or distribute the Licensed Source Code
apart from the executable form incorporated in or otherwise as a unit within the
Licensed Products.
(b) Notwithstanding any other provision of this Agreement,
including without limitation the provisions of Article V regarding Improvements,
all modifications to or other derivative works of the Licensed Source Code made
by Licensee pursuant to the foregoing Section 2.3(a) shall be owned by Licensee.
Subject to the terms and conditions of this Agreement, Licensee hereby grants to
Licensor a non-exclusive, royalty-free worldwide license to such modifications
and derivative works solely for the internal use of Licensor within the Field of
Use. Licensee further grants to Licensor a non-exclusive, worldwide license to
sublicense such modifications and derivative works to third parties for use
outside the Field of Use, upon terms agreed upon in an amendment hereto, which
shall include provisions for a reasonable royalty to Licensee as will be set
forth on a schedule attached hereto. Licensee does not otherwise grant any
rights to Licensor to use or sublicense for use such modifications and
derivative works outside the Field of Use. Upon the development of any such
modification or derivative work, Licensee shall provide to Licensor in both
Source Code and compiled object code formats, a copy of the revised Licensed
Software, together with adequate documentation and information to enable a
person of reasonable skill in the relevant programming language(s) to understand
and maintain the Licensed Software as modified by Licensee.
2.4 Licensee agrees to reproduce and apply any copyright, patent,
trademark or other proprietary rights notices to all copies of the Licensed
Product as requested by Licensor.
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ARTICLE III
PAYMENTS
3.1 License Fee.
(a) Licensee agrees to pay to Licensor a license fee,
allocated according to the following ratio [**] divided between Licensed
Patents, Licensed Patent Applications, and Licensed Software respectively, in
accordance with the following schedule:
(1) Three Hundred Thousand Dollars ($300,000)
simultaneously with the execution of this Agreement.
(2) [**] on the first anniversary of the Effective
Date. Licensee agrees to deliver to Licensor by the first anniversary of the
Effective Date Four Hundred Thousand Dollars ($400,000) worth of Licensee's
products selected by Licensor, the value of which shall be determined using
prices which are generally offered to commercial purchasers of such products in
similar quantities.
(3) [**] on the second anniversary of the Effective
Date.
(4) [**] on the third anniversary of the Effective
Date.
(b) The obligation of Licensee to pay said license fees shall
immediately terminate upon the termination of Licensor's right to license the
Licensed Patents subject to Section 10.2(d).
(c) Payment of the license fee shall be made by wire transfer
to the account of the Licensor specified in Section 3.2(c) hereof or to such
other account as may be specified in a notice to Licensee not later than two
business days prior to the due date of such payment.
3.2 Royalty Payment.
(a) Commencing on the Effective Date, Licensee shall pay to
licensor a royalty on the Licensed Products of [**] of Net Sales of all
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Licensed Products covered by a Subsisting Claim and containing Licensed
Software; provided, however, that in the event that Licensee is required to pay
a royalty to any third party for an element of the Licensed Product covered by
one or more Subsisting Claims and in order to make, use or sell a Licensed
Product, Licensor shall reduce the total fees payable to Licensor or Licensor's
Affiliate hereunder or pursuant to any other arrangement relating to the sale of
the Licensed Products (collectively, the "Total Payments") by one half of the
amount of the royalty paid to such third party. In the event a Licensed Product
is not covered by a Subsisting Claim, the royalty shall be [**] of Net Sales of
all such Licensed Products. In the event a Licensed Product does not contain
Licensed Software, the royalty shall be [**] of Net Sales of all such Licensed
Products. The royalty payments shall be subject to the same ratio applied in
Section 3.1 herein, where the portion of the payment pertaining to Licensed
Patents shall persist as long as there is a Subsisting Claim.
(b) Licensee shall pay to Licensor within thirty (30) calendar
days after the last day of each calendar quarter during the Term (each a
"Royalty Period") the respective royalty payable to Licensor pursuant to this
Section 3.2. Each royalty payment shall be made by wire transfer to the account
of Licensor at West America or to such other account specified in a notice to
Licensee prior to the last day of the applicable Royalty Period. Simultaneously
with each royalty payment, Licensee shall furnish to Licensor a report showing
(i) the Net Sales of the Licensed Products during the Royalty Period then ended
and (ii) the calculation of the royalties payable for such Royalty Period.
3.3 Books and Records. Licensee agrees to maintain accurate and
complete books and records concerning the sale of Licensed Products and the
calculation of Net Sales. Licensee shall permit Licensor or its authorized
agents to inspect such books and records at Licensee's facilities upon
reasonable notice during regular business hours during the Term. Licensee agrees
to maintain accurate and complete books and records concerning all copies made
of the Licensed Source Code and all persons who see or access the Licensed
Source Code or any copy thereof. Such books and records shall include a record
of each copy made and destroyed, a description of
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each change made to the Licensed Source Code, and all parties involved in making
such change, and the relevant dates for all of the foregoing.
3.4 Calculation Dispute. The parties shall endeavor to resolve promptly
any dispute regarding the calculation of the amounts payable or expended under
Sections 3.2, 9.2 and 10.2(c). If the parties are unable to resolve any such
dispute within ninety (90) calendar days of the day that such disputed payment
would be payable hereunder, then the Licensor and Licensee shall submit the
items in dispute for resolution to Ernst & Young, and each party shall advise
such firm in writing of its position regarding such dispute. Ernst & Young shall
be instructed to determine such disputed items in accordance with the terms of
this Agreement and issue its determination in writing to both parties together
with the reasons therefor within thirty (30) calendar days after submission.
Such determination shall be final and binding on both parties and the parties
agree to promptly make appropriate payments in accordance with such
determination. All royalties or license fees due within this time period shall
be held in escrow and shall accrue reasonable interest. If the determination
results in a finding that Licensee underpaid more than or equal to ten percent
(10%) of the established amount, Licensee shall pay the fees and disbursements
of Ernst & Young. If the determination results in a finding that Licensee
underpaid less than ten percent (10%), the fees and disbursements of Ernst &
Young shall be allocated equally between the Licensor and the Licensee. The
choice of Ernst & Young as the accounting firm is subject to its lack of
representation of either party at the time of the Calculation Dispute. Should
Ernst & Young represent either party at that time, the parties shall mutually
appoint a "big six" independent certified public accounting firm.
3.5 Exclusions from Royalty. No royalty shall be payable on any
Licensed Products used by Licensee, its Affiliates or sublicensees for testing,
research and development in connection with exercising the licenses granted
herein, or solely for other internal, non-commercial purposes, including,
without limitation, training, demonstration and marketing.
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ARTICLE IV
DOCUMENTATION
4.1 Documentation. As promptly as practicable after the execution of
this Agreement, Licensor shall deliver to Licensee, as reasonably required to
exercise the rights granted hereunder, documentation in writing concerning the
practice and use of the Intellectual Property and Improvements thereto. Such
documentation shall include the Licensed Source Code and any technical drawings,
manuals or other materials concerning the design, development, manufacture
and/or use of the Licensed Patents, Licensed Patent Applications, Proprietary
Information and Licensed Software. Licensor represents and warrants that the
documentation furnished to Licensee under this Section 4.1 shall be complete,
accurate and sufficient to permit a person reasonably skilled in the relevant
art to practice the Intellectual Property in the Field of Use as contemplated by
this Agreement. Such documentation shall be considered Confidential Information
and shall be kept confidential in accordance with Section 9.4 herein.
ARTICLE V
IMPROVEMENTS
5.1 Improvements by Licensor. In the event that Licensor shall make any
Improvements during the Term, Licensor shall promptly disclose in writing the
same to Licensee subject to the confidentiality provisions of this Agreement and
such Improvement shall be deemed to be included within the Intellectual Property
licensed hereunder; provided, however, that in the event that Licensor shall
secure the grant of Letters Patent on any such Improvements, it shall so notify
Licensee and, if Licensee elects to be licensed under such patent, Licensor
shall include such patent within the Licensed Patents upon the terms agreed upon
herein, which shall include provisions for a reasonable royalty to Licensor and
will be set forth on a schedule attached hereto. Any patents which may issue
from the Licensed Patent Applications shall not be subject to this Section 5.1.
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5.2 In the event that Licensee shall make any Improvements during the
Term other than modifications to or derivative works of the Licensed Source Code
which shall be controlled by the provisions of Section 2.3, Licensee shall
promptly disclose the same to Licensor subject to the confidentiality provisions
of this Agreement. Licensee grants and hereby does grant to Licensor a license
for such Improvement on a non-exclusive, royalty-free worldwide basis for uses
outside the Field of Use, upon the terms agreed upon herein, excluding royalty
payments.
5.3 Notification of each Improvement shall be made promptly, but in any
event, no later than the quarterly reporting period after the reduction to
practice or the acquisition of such an Improvement which is already reduced to
practice, and shall include a disclosure, in written or other tangible form
reasonably acceptable to the party receiving such disclosure and sufficient to
allow such party to practice the Improvement.
ARTICLE VI
INFRINGEMENT
6.1 Infringement Notice. During the Term, if either party becomes aware
that a Person is Infringing any Intellectual Property in the Field of Use, such
party shall promptly notify the other party thereof. Such notice shall set
forth, to the extent known, the identity of the alleged Infringer, the nature of
the Infringement, the date and location of the same, and any other information
such party may have regarding the Infringement.
6.2 Commencement of Action.
(a) Licensor represents and warrants that to the best of
Licensor's knowledge no Person is Infringing any of the Intellectual Property as
of the date of this Agreement.
(b) Not more than one hundred and twenty (120) calendar days
after it becomes aware of any Infringement of the Intellectual Property in the
Field of Use, whether by notice from Licensee or otherwise, Licensor shall
commence and diligently prosecute an action for infringement and/or
misappropriation against the alleged Infringer; provided, however, that Licensor
may, in its sole discretion, extend said one hundred and twenty (120) day period
for up
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to sixty (60) additional calendar days for the purpose of making good faith
efforts to resolve the situation with the alleged Infringer. Licensor shall be
entitled to use its reasonable discretion in determining whether to pursue an
action for infringement, and such reasonable discretion may include reliance on
opinions by counsel.
(c) At Licensor's reasonable discretion, Licensor shall
furnish to Licensee information and materials disclosing material developments
in any action so commenced by Licensor (or in any declaratory judgment action
commenced by the alleged Infringer).
(d) If Licensor decides not to commence an action as specified
in Section 6.2(b) hereof, Licensee may, upon approval of Licensor not to be
unreasonably withheld or delayed, commence such action in its own name and at
its own expense, and may join Licensor as a party plaintiff or as a co-plaintiff
or, in any declaratory judgment action the Infringer may commence, as a
co-defendant. The right of Licensor to withhold approval of commencement of such
action by Licensee is subject to advice by counsel that such suit would not be
advisable. If any such action is commenced properly by Licensee pursuant to this
Section 6.2(d) then the following shall govern:
i) Licensee shall have the right to settle any such
claim, suit or proceeding upon written consent of Licensor, such consent not to
be unreasonably withheld or delayed;
ii) If damages or settlement amounts are received,
Licensee's costs will be reimbursed and if Licensor was joined and incurred
costs associated with the litigation, said costs will also be reimbursed; and
iii) If, after reimbursing Licensee and Licensor, if
necessary, there are remaining damages or settlement amounts, these will be
allocated evenly (50:50) between Licensor and Licensee.
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6.3 Indemnity by Licensor for Intellectual Property Infringement.
(a) Licensor represents and warrants to Licensee that (i) it
holds all proprietary right, title and interest in and to the Licensed Software,
(ii) it has the right to license to Licensee the Licensed Patents in the manner
set forth herein and, (iii) as of the Effective Date, Licensor has not been put
on notice that the use of the Intellectual Property by Licensee as contemplated
by this Agreement constitutes an Infringement. At its expense, Licensor shall
defend or settle any claim, suit or proceeding brought against Licensee by a
Person claiming Infringement based on the licensed use of the Intellectual
Property otherwise in accordance with this Agreement. In addition, Licensor
shall indemnify and hold harmless Licensee from any Liabilities resulting from
an adjudication of any such claim, suit or proceeding in which it is determined
that Licensee's practice of the Intellectual Property hereunder in compliance
with the terms and conditions of the Agreement constitutes Infringement. At the
option of Licensor, Licensee may participate, at the expense of the Licensee, in
the defense of any such claim, suit or proceeding. Licensor shall retain control
of the proceeding and shall, at its reasonable discretion, furnish to Licensee
copies of all material papers filed in the proceeding.
(b) As a condition precedent to Licensor's obligations under
this Section 6.3, Licensee shall (i) give Licensor notice of the written threat
of any such claim, suit or proceeding or the written assertion by any Person
that Licensee's use of the Intellectual Property constitutes an Infringement of
such Person's intellectual property rights, and (ii) reasonably cooperate with
Licensor in respect to any defense of such claim, suit or proceeding. Licensor
shall control the defense of any such legal action and may rely on opinions of
counsel. Licensor shall have the right to settle any such claim, suit or
proceeding without the consent of Licensee, provided, however, that in the event
any such settlement would cause Licensee to incur substantial liability or
obligation, financial or otherwise, or to be restricted from the practice of the
Intellectual Property as contemplated by this Agreement, such settlement shall
require the prior written consent of Licensee, which consent shall not be
unreasonably withheld.
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(c) The foregoing indemnity shall not extend to any claim of
infringement resulting from any modification of the Intellectual Property not
furnished by Licensor, including without limitation any modification of or
derivative work of the Licensed Source Code or the combination of the
Intellectual Property with any other technology or intellectual property not
provided by Licensor hereunder. In addition, at its expense, Licensee shall
indemnify and hold harmless Licensor from any Liabilities arising from an
adjudication or settlement of any claim, suit or proceeding in which it is
alleged or determined that any modifications of the Licensed Source Code
constitute Infringement.
ARTICLE VII
LIMITATION OF REMEDIES
7.1 Except for the termination penalties set forth in Section 10.2
hereof and any Liabilities covered by Section 6.3, neither party to this
Agreement shall be liable for any incidental, consequential or special damages
of any kind, whether arising in tort, contract or otherwise, and regardless of
whether a party had been advised of the possibility of such damages.
ARTICLE VIII
REPRESENTATIONS AND WARRANTIES
8.1 Representations and Warranties of Licensor. Licensor represents and
warrants to Licensee that (i) it is a corporation organized and existing under
the laws of the State of Delaware, (ii) it has full right, power and authority
to execute and perform this Agreement, (iii) the execution and performance of
this Agreement do not and will not violate any law, rule, regulation, order,
writ, injunction or decree of any court or government, domestic or foreign, or
any commission, bureau or administrative agency, or any agreement or instrument
by which Licensor is bound, and (iv) upon its execution, this Agreement will
constitute the binding obligation of Licensor enforceable against it in
accordance with its terms, except as the same may be limited by bankruptcy,
insolvency, reorganization or other laws relating to or affecting
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the enforcement of creditors' rights generally or by limitations on the
availability of equitable remedies.
8.2 The warranties set forth herein are exclusive and in lieu of all
other warranties express or implied, including, without limitation, the implied
warranties of merchantability and fitness for a particular purpose. Licensor
neither assumes, nor authorizes any person to assume for it, any other liability
in connection with the Licensed Product, including, without limitation,
liability arising out of the delivery or use of the Licensed Product.
8.3 Representations and Warranties of Licensee. Licensee represents and
warrants to Licensor that (i) it is a corporation organized and existing under
the laws of the State of New York, (ii) it has full right, power and authority
to execute and perform this Agreement, (iii) the execution and performance of
this Agreement do not and will not violate any law, rule, regulation, order,
writ, injunction or decree of any court or government, domestic or foreign, or
any commission, bureau or administrative agency, or any agreement or instrument
by which Licensee is bound, and (iv) upon its execution, this Agreement will
constitute the binding obligation of Licensee enforceable against it in
accordance with its terms, except as the same may be limited by bankruptcy,
insolvency, reorganization or other laws relating to or affecting the
enforcement of creditors' rights generally or by limitations on the availability
of equitable remedies.
ARTICLE IX
ADDITIONAL OBLIGATIONS OF THE PARTIES
9.1 Performance under the Ohio State License Agreement. During the
Term, Licensor shall use its best efforts to maintain in full force and effect
the Ohio State License Agreement and shall perform all of its obligations
thereunder. In furtherance of the foregoing and not by way of limitation,
Licensor shall, among other things, timely pay all license fees and royalty
payments under such agreement. Licensor shall promptly provide Licensee with
copies of any and all notices or other communications to or from Licensor
relating to the Ohio State
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License Agreement and shall not amend, modify or terminate the Ohio State
License Agreement without the prior written consent of Licensee which consent
shall not be unreasonably withheld. Licensor shall provide Licensee with written
notice within sixty (60) days prior to the obligation to meet a payment under
the Ohio State License if Licensor does not intend to meet the obligation. At
its discretion upon receipt of such notice, Licensee may meet the obligation,
and any such expenditures shall be offset against any future royalties or
license fees due to Licensor under this Agreement. Upon loss by Licensor of the
right to license the Licensed Patents, Licensee may then pursue an agreement
with OSU in accordance with the terms of Exhibit A attached hereto.
9.2 Research and Development. Licensee agrees to commit at least [**]
per year, for at least two years following the Effective Date, for research and
development related to the commercial exploitation of fluorescence imaging
systems for use in the Field of Use.
9.3 Announcement of Agreement. The parties agree that any public
announcement of this Agreement may make reference to the terms of Section 9.2
and to the fact that royalty provisions are included in the Agreement without
stating the details of such provisions. The parties further agree that each
shall make copies of the public announcements available to the other in
sufficient time to allow review by counsel. Release of the announcements by one
party shall be subject to the approval of the other party, such approval not to
be unreasonably withheld.
9.4 Confidentiality. Licensee acknowledges that the Confidential
Information and Proprietary Information disclosed by Licensor pursuant to this
Agreement is the confidential information of Licensor. Licensee agrees that it
shall not use the Confidential Information or Proprietary Information except as
is necessary for the exercise of the license granted in this Agreement, nor
shall Licensee disclose the Confidential Information or Proprietary Information
to any third party (other than subcontractors approved by Licensor who agree not
to disclose or use the Confidential Information or Proprietary Information
except as necessary in the exercise of a sublicense permitted by this Agreement
and excluding Licensed Source Code) without the
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prior written consent of Licensor. The parties acknowledge and agree that the
foregoing restrictions shall not apply to any information which:
(a) was in the public domain at the time of Licensor's
disclosure to Licensee;
(b) entered the public domain through no fault of Licensee
subsequent to the time of Licensor's disclosure to Licensee;
(c) was in Licensee's possession free of any obligation of
confidence at the time of Licensor's disclosure to Licensee; or
(d) was developed by Licensee independently of and without
reference to the Proprietary Information.
9.5 Licensed Source Code Confidentiality. Without limiting the
foregoing, and without limiting the restrictions on Licensee included in the
provisions of Section 2.2 hereof, Licensee acknowledges that the Licensed Source
Code constitutes a valuable trade secret of Licensor; and shall protect the
confidentiality of the Licensed Source Code in a manner at least as protective
as that used by Licensee to protect its own similar trade secrets and source
code.
9.6 Reversion of Rights
(a) For purposes of this Section 9.6, the phrase "Reversionary
Field of Use" shall mean all uses of the Intellectual Property within the Field
of Use, where "hybridization" shall include any binding event such as nucleic
acid hybridization and protein/protein hybridization in conjunction with
fluorescence emission and absorbance determinations. Hybridization based assays
include, without limitation, slide-based cytometry, analysis of arrays of
biomolecules on slides, and analysis of hybridization occurring on spots or on
xxxxx in slides or plates. The Reversionary Field of Use shall include
non-hybridization-based assays including, without limitation, clinical
diagnostics, gel scanning, thin layer chromatography scanning and
electrophoresis-based nucleic acid sequencing.
(b) Licensee and Licensor hereby agree that if Licensee
decides not to exploit or to cease exploiting the Intellectual Property in all
or part of the Reversionary Field of Use, Licensee shall so inform Licensor in
the quarterly royalty report issued immediately following
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the making of such decision. Upon the receipt of such written notification,
Licensor shall have 30 (thirty) days to inform Licensee in writing if Licensor
has decided to exploit the Intellectual Property for such use or uses. If
Licensor does not provide timely written notice, then all rights in this
Intellectual Property for such use(s) shall remain with Licensee, to the extent
they are granted to Licensee in this Agreement. If Licensor provides timely
written notice, Licensee's right to exploit the Intellectual Property for such
use(s) shall terminate, and such use(s) shall be excluded from the Field of Use
for the remaining term of this Agreement. All such terminated rights shall
revert to Licensor, and Licensor shall have unrestricted rights (as against
Licensee) in and to the Intellectual Property in such part of the Reversionary
Field of Use.
ARTICLE X
TERM AND TERMINATION
10.1 Term. Unless otherwise renewed or terminated as provided in
Section 10.2 below, this Agreement shall commence on the Effective Date and
shall continue for so long as there exists a Subsisting Claim in any of the
Licensed Patents or the Licensed Software is included in a Licensed Product.
10.2 Termination. This Agreement may be terminated by the parties as
follows:
(a) The parties may terminate this Agreement at any time by
mutual written consent.
(b) Either party may terminate this Agreement upon thirty (30)
calendar days prior notice if the other party shall commit a material breach of
this Agreement and such breach shall not be cured by such other party within
thirty (30) calendar days of receipt of such notice.
(c) Subject to the following termination penalty, Licensee may
terminate this Agreement at any time upon thirty (30) calendar days prior notice
upon payment of the termination penalty. If Licensee elects to terminate at any
time within the first nine (9) months following the Effective Date, Licensee
shall pay no penalty provided that the anticipated research and development fees
under Section 9.2 have been spent. At the nine month date, the anticipated
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expenditures are 9/12 of [**] or [**] or a pro rata share of such amount as of
an earlier termination within this nine (9) month period (e.g., [**] at six (6)
months). Should Licensee fail to spend at least [**] within this time period or,
such pro rata amount, Licensee will pay Licensor fifty percent (50%) of the cash
amount set forth in Section 3.1(a)(2). If Licensee terminates the agreement
after the ninth (9th) but before the end of the twelfth (12th) month following
the Effective Date, Licensee shall pay seventy-five percent (75%) of the cash
amount set forth in Section 3.1(a)(2). The termination penalty after this period
shall be on a pro rata basis calculated as the equation (1-(X/[**]))Y where X is
the amount of actual expenditures under section 9.2 and Y is the amount set
forth in Section 3.1(a)(3).
(d) Upon payment in full of all outstanding license fees and
royalty payments due and payable, Licensee may terminate this Agreement at any
time upon the termination of Licensor's right to license the Licensed Patents.
10.3 Consequences of Termination; Expiration. Upon the termination of
this Agreement:
(a) Any amounts due and payable to either party hereunder as
of the date of termination shall continue to be payable in accordance with the
terms hereof.
(b) Licensee shall cease the manufacturing and sale of the
Licensed Products, except for a limited period as necessary to sell off its
work-in-process and finished inventory of Licensed Products (the "Sell-Off
Period"), as of the date of the notification, such Sell-Off Period not to exceed
six months. There shall be no Sell-Off-Period if Licensor's right to license the
Licensed Patents expires or is otherwise terminated or if Licensor no longer has
no right to license the Intellectual Property.
(c) The licenses granted to Licensee hereunder shall terminate
except that a license to sell shall continue for the duration of the Sell-Off
Period.
(d) Licensee shall return to Licensor or certify in writing
the destruction of all original materials and copies thereof that embody the
Proprietary Information including without limitation all copies of the original
Licensed Source Code and all documentation relating thereto.
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10.4 Survival. Licensee's obligation to maintain the confidentiality of
the Proprietary Information shall survive any termination of this Agreement. The
indemnity obligations of Licensor pursuant to Article VI and the obligations of
the parties pursuant to this Article X shall also survive any termination or
expiration of this Agreement.
10.5 Suspension of Performance. If Licensor shall commit a material
breach and not cure such breach within thirty (30) days of Notice, in accordance
with Section 10.2(b), Licensee shall have the option of (i) exercising its right
to terminate this Agreement pursuant to Section 10.2(b) or (ii) notifying
Licensor of such breach and suspending performance of all its duties hereunder
(excluding payment of licensee fees and royalties as otherwise required by
Sections 3.1 and 3.2 hereof) until such breach is cured. In the event of a
dispute, other than a dispute under Section 3.4, in which Licensee in good faith
asserts that Licensor's alleged breach warrants a suspension of royalty payments
and license fees to Licensor under Section 3.2, Licensee shall have the right to
pay all amounts payable to Licensor under Section 3.2 into a third-party escrow
account during the pendency of such dispute, provided that all escrowed funds
remain in such escrow account until resolution of the dispute by an arbitrator
pursuant to Section 11.3 (the "Escrow Period"). Licensee shall continue to be
obligated by all other terms and conditions of this Agreement except as
otherwise set forth in this Section 10.5 during any such Escrow Period.
Licensee's suspension of the performance of its obligations pursuant to this
Section 10.5 shall not constitute a breach of this Agreement. Nothing in this
Section 10.5 shall limit the right of Licensee to seek such other remedies as
may be available to it under this Agreement or otherwise by reason of any such
breach of Licensor.
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ARTICLE XI
MISCELLANEOUS
11.1 Notices. Any notice or other communication provided for in this
Agreement shall be in writing and shall be delivered personally, or sent by
facsimile transmission or nationally recognized overnight courier service, and
shall be deemed given when so delivered personally or by facsimile transmission,
with receipt confirmed, or one (1) business day after the date of deposit with
such courier service. All notices and other communications shall be addressed to
the parties at their respective addresses as set forth below. Each party hereto
may change its address upon written notice to the other party in the manner
provided in this Section 11.1.
If to Licensor:
LXR Biotechnology Inc.
0000 Xxxxxx Xxx Xxxxx
Xxxxxxxx, XX 00000
Facsimile No.: (000) 000 0000
If to Licensee:
The Xxxxxx-Xxxxx Corporation
000 Xxxx Xxxxxx
Xxxxxxx, XX 00000-0000
Attention: Secretary
Facsimile No.: (000) 000-0000
11.2 Governing Law. This Agreement shall be governed by, and construed
and enforced in accordance with, the laws of the State of New York without
giving effect to its rules governing choice of law.
11.3 Arbitration; Injunctions. Subject to Section 3.4 hereof, all
disputes, controversies or differences between the parties which may arise out
of, in relation to, or in connection with this Agreement shall, unless settled
by mutual consultation in good faith, be settled by arbitration in New York in
accordance with the Commercial Arbitration Rules then in effect of the American
Arbitration Association, and judgment upon the award rendered by the arbitrators
may
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be entered in any court having jurisdiction thereof. Prior to filing for
Arbitration, the parties will set aside a "cool-off" period of thirty (30) days
during which senior management of the parties will meet and attempt to resolve
any disputes amicably. Notwithstanding anything to the contrary contained in
this Section 11.3, each party reserves the right to pursue injunctive relief or
other equitable remedies in a court of competent jurisdiction in connection with
any breach of the terms of this Agreement.
11.4 Attorneys' Fees. If any action at law or in equity, including
action for arbitration or injunctive relief, is brought relating to this
Agreement or the breach thereof, the prevailing party in any final judgment or
arbitration award, or the non-dismissing party in the event of a dismissal
without prejudice, shall be entitled to the full amount of all reasonable
expenses, including all court costs, arbitration fees and actual attorneys' fees
paid or incurred in good faith.
11.5 Assignment. Subject to Section 2.2 hereof, this Agreement may not
be assigned by either party, except that either party may assign all of its
rights and delegate all of its obligations hereunder to an Affiliate or to any
third party that is a successor to all or substantially all of the assets and
business operations of such party and either party may assign all of its rights
and delegate all of its obligations hereunder to a Person acquiring all of the
assigning party's rights to the Intellectual Property within all or a portion of
the Field of Use upon prior written notice and subject to approval by the other
party which approval will not be unreasonably withheld. This Agreement shall be
binding upon and inure to the benefit of the parties and their respective
successors and permitted assigns.
11.6 Force Majeure. Neither party to this Agreement will be liable for
failure to perform any of its obligations hereunder during any period in which
such performance is delayed by acts beyond its reasonable control, including,
without limitation, fire, flood, war, riot, embargo, organized labor stoppage,
earthquake, and acts of civil and military authorities, provided that the party
suffering such delay immediately notifies the other party of the delay, and,
further, that either party shall have the right to terminate this Agreement upon
ninety (90)
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days prior written notice if the delay of the other party due to any of the
above-mentioned causes continues for a period of four (4) months.
11.7 Severability. In the event that a provision, or part thereof, of
this Agreement shall be held to be invalid, illegal or unenforceable, the
validity, legality and enforceability of the remaining provisions, or parts
thereof, shall not in any way be affected or impaired thereby.
11.8 Waiver. No waiver of any breach of this Agreement shall be
effective unless made in writing, and no waiver shall constitute a waiver of any
subsequent breach.
11.9 Relationship between Parties. No license is granted hereunder to
any party other than as specifically set forth herein. The relationship
established by this Agreement is solely that of a licensor and licensee and
nothing contained herein shall create a partnership, joint venture or any other
business relationship between Licensor and Licensee. Except as expressly set
forth herein, neither party shall have authority to obligate or bind the other
party with respect to any matter, to make any contract, sale, agreement,
warranty or representation, express or implied, on behalf of the other party, or
to accept any legal process for or on behalf of the other party.
11.10 Headings; References. The Article and Section headings contained
herein are for reference purposes only and shall not in any way affect the
meaning or interpretation of this Agreement.
11.11 Entire Agreement. This Agreement constitutes the entire agreement
and understanding between the parties hereto with respect to the subject matter
hereof and supersedes all prior discussions, agreements, and understandings of
any and every nature, whether written or oral, with respect to the same matter.
11.12 Amendments. No amendments, modifications or supplements to this
Agreement shall be enforceable or binding upon the parties hereto unless made in
a writing expressly referring to this Agreement and executed by a duly
authorized representative or officer of each of the parties hereto.
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11.13 Counterparts. This Agreement may be executed in one or more
counterparts, each of which when executed shall be deemed to be an original, but
all of which taken together shall constitute one and the same instrument. This
Agreement may be executed via facsimile signature.
IN WITNESS WHEREOF, the parties hereto have caused this Agreement to be
executed as of the day and year first written above.
LXR BIOTECHNOLOGY INC.
By: /s/L. Xxxxx Xxxxx
-----------------------------------
Name: L. Xxxxx Xxxxx
Title: LXR Biotechnology Inc.
President
THE XXXXXX-XXXXX CORPORATION
By: /s/Xxxx X. Xxxxxx
-----------------------------------
Name: Xxxx X. Xxxxxx
Title: Senior Vice President Corporate
Development and
Chief Technology Officer
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SCHEDULE 1.12
Licensed Patents
U.S. Patent No. 5,037,207 to Tomei et al.
U.S. Patent No. 4,877,966 to Tomei et al.
U.S. Patent No. 4,758,727 to Tomei et al.
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SCHEDULE 1.13
Licensed Patent Applications
1. U.S. Serial No. 08/451,325 "Multi-Channel Acquisition Using Integrating
Sphere," filed May 26, 1995.
2. WO/US96/06808 PCT of 08/451,325, filed May 13, 1996.
3. U.S. Serial No. 08/452,035, "Wide Angle Scattering Detector," filed May
26, 1995.
4. WO/US96/07374 PCT of 08/452,035, filed May 21, 1996.
5. [**]
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SCHEDULE 1.15
A. Licensed Software
[**]
B. Licensed Source Code
[**]
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SCHEDULE 1.19
DESIGNS OF ALL MECHANIC COMPONENTS, CONSISTING OF:
[**]
DRAWINGS OF ELECTRONIC AND ELECTRICAL WIRING, CONSISTING OF: (16)
[**]
INFORMATION ON ALL OTHER (COMMERCIAL) PARTS AND PARTS VENDORS
PERFORMANCE ANALYSIS ON SYSTEM (VIBRATION) ISOLATION
PERFORMANCE ANALYSIS ON OPTICAL FOCUSING SYSTEM
INSTRUMENTATION CONCERNING HARDWARE AND FIRMWARE, CONSISTING OF:
[**]
INSTRUMENTATION CONCERNING SOFTWARE, CONSISTING OF:
[**]
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[**]
COMPLETE DOCUMENTATION INCLUDING:
[**]
TECHNOLOGY (OR PRODUCT) CURRENTLY UNDER DEVELOPMENT:
[**]
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CONFIDENTIAL TREATMENT REQUESTED
LXR BIOTECHNOLOGY INC.
LOCATED AT 0000 XXXXXX XXX XXXXX
XXXXXXXX, XXXXXXXXXX 00000
XXXXXXXX & XXXXXXXX LLP
SAN FRANCISCO ATTORNEYS AT LAW NEW YORK
LOS ANGELES WASHINGTON, D.C.
SACRAMENTO 000 XXXX XXXX XXXX XXXXXX
XXXXXX XXXXXX PALO ALTO, CALIFORNIA 94304-1018 BRUSSELS
WALNUT CREEK TELEPHONE (000) 000-0000 HONG KONG
SEATTLE TELEFACSIMILE (000) 000-0000 TOKYO
DENVER
EXHIBITS
TO
LICENSE AGREEMENT
LICENSE AGREEMENT dated as of August 29, 1996, between, LXR BIOTECHNOLOGY INC.,
a Delaware corporation, on behalf of itself and OPTICAL ANALYTIC, INC., its
wholly-owned subsidiary ("Licensor"), and THE XXXXXX-XXXXX CORPORATION, a New
York corporation ("Licensee").
CONFIDENTIAL
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** CONFIDENTIAL TREATMENT REQUESTED
TABLE OF CONTENTS
CONFIDENTIAL
A. Letter of August 27, 1996 from Ohio State University to Xxxxxx-Xxxxx .......
B. Ohio State University/LXR License and Modifications ........................
1. Letter of August 15, 1991 from Ohio State to Optical Analytic, Inc.
regarding Cooperative Agreement, with Agreement attached. .........
2. Letter of November 23, 1993 from Optical Analytic, Inc. to LXR
regarding extension of Milestones. ................................
3. Letter of November 29, 1993 from Ohio State University to
Optical Analytic, Inc. regarding extension of Milestones. .........
4. Amendment to Agreement of July 10, 1996, deleting
Milestone paragraphs ..............................................
5. Unexecuted Amendment to Agreement, deleting patent No. 4,601,537 ..
C. Patents and Applications. ..................................................
1. U.S. Patent No. 5,037,207 to Tomei et al. .........................
2. U.S. Patent No. 4,877,966 to Tomei et al. .........................
3. U.S. Patent No. 4,758,727 to Tomei et al. .........................
4. U.S. Serial No. 08/451,325 ........................................
"Multi-Channel Acquisition Using Integration Sphere," filed
May 26, 1995 (WO/US96/06808 PCT of 08/451,325, Filed May 13,
1996)
5. U.S. Serial No. 08/452,035 ........................................
"Wide Angle Scattering Detector," filed May 26, 1995
(WO/US96/07374 PCT of 08/452,035, Filed May 21, 1996)
6. **[" "]....
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Exhibit A to Xxxxxx-Xxxxx License Agreement
33
------------------------
T . H . E Office of Technology Transfer 0000 Xxxxx Xxxx
XXXX Xxxxxxxx, XX 00000-0000
STATE
UNIVERSITY Phone 000-000-0000
------------------------
August 27, 1996
Xxxxxxx X. Xxxxxxxx
Xxxxxx-Xxxxx Corporation
000 Xxxx Xxxxxx
Xxxxxxx, XX 00000-0000
Subject: License Agreement for Scanning Laser Imaging Technology.
Dear Xx. Xxxxxxxx:
I understand that Xxxxxx-Xxxxx (PE) is negotiating a license with LXR
Biotechnology Inc. through its wholly owned subsidiary Optical Analytic, Inc.
(OA) a license for the subject technology in the field of Research and Clinical
Applications. The technology was exclusively licensed to OA by The Ohio State
University and The Ohio State University Research Foundation (collectively OSU)
in an agreement dated August 15, 1991, which has been amended and now covers the
intellectual property in U.S. Patents # 4,758,727; 4,877,966 and 5,037,207.
This letter is to assure PE that OSU will negotiate a similar exclusive license
containing the same conditions of diligence, minimum royalties, liability etc.
as contained in the OA agreement should the agreement with OA (LXR) be
terminated.
Please let me know if you have any further questions.
Regards,
/s/ Xxxx X. Lafayatis
---------------------
Xxxx X. Lafayatis, Director Technology Transfer
cc: X.X. Xxxxxxxxxx, OSU
X.X. Xxxxx, LXR
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Exhibit B1 to Xxxxxx-Xxxxx License Agreement
35
------------------------
T . H . E Office of Technology Transfer 0000 Xxxxx Xxxx
XXXX Xxxxxxxx, XX 00000-0000
STATE
UNIVERSITY Phone 000-000-0000
------------------------
August 15, 1991
F. Xxxxx Xxxxx
Optical Analytic, Inc.
0000 Xxxxxxxxx Xxxxx
Xxxxxx, XX 00000
Subject: SLI license
Dear Xx. Xxxxx:
I enclose three counterparts of the final license agreement for the SLI
technology that you approved in draft. These have been executed by the
University and the Research Foundation. Please have them executed by OAI and
return two to me.
Pursuant to our phone conversation earlier this week, if OAI and Xxxxxx, Inc. go
forward with a joint venture (or a merger or any other cooperative arrangement)
for the commercial development of the SLI technology, we would expect that our
rights and interests under each of your primary co-exclusive license agreements
would be fully protected and that all of the obligations of the licensee under
each of those primary license agreements would be cumulatively fulfilled, with
any exceptions to be only with our express written consent. For example, we
would expect that the total royalties payable to us under both primary license
agreements for any period would never be less than the sum of the minimum
royalties specified in those two agreements for that period, i.e. that no
royalty amount accrued in respect of any transaction would be credited against
the minimum royalties specified in both primary license agreements and that no
minimum royalty paid or payable under one primary license agreement would be
credited against royalties paid or payable under the other.
On the other hand, we would not expect that duplicate royalties would be paid or
payable under both primary licenses in respect of any particular unit of
Licensed Product or Service merely because the royalty-generating transaction
involving such unit might technically be made under the authority of both
primary licenses rather than only under the authority of one of them.
Furthermore, to cover all of the hypothetical possibilities, in case there
should be a joint or cooperative transaction not solely attributable to either
primary licensee, and in respect of which the two primary license agreements
called for different royalty payments to us, we would expect
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that an undivided half of such transaction would be deemed to have been subject
to each of the primary license agreements.
Finally, we construe the provisions of 8.2 of the OAI license agreement as
creating both obligations of OAI and rights of OSURF respecting activities
undertaken by joint ventures in which OAI has an interest, or by other joint
venture "partners" for the benefit of such a joint venture.
It would probably be best to formalize these understandings among all interested
parties when actual arrangements for any joint venture or other cooperation have
been worked out.
Sincerely,
/s/ Xxxxx X. Xxxxxxx
--------------------
Xxxxx X. Xxxxxxx
Patent and Copyright Administrator
Encl. - 3 partially executed counterparts of "oaisli.lc4"
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oaisli.lc4
LICENSE AGREEMENT
This License Agreement is entered into between Optical Analytic, Inc., ("OAI"),
a corporation of Ohio having a principal place of business at 0000 Xxxxxxxxx
Xx., Xxxxxx, Xxxx 00000, and The Ohio State University Research Foundation
("OSURF"), a non-profit corporation of Ohio having a principal place of business
at 0000 Xxxxx Xxxx, Xxxxxxxx, Xxxx 00000-0000 and acting on behalf of itself and
The Ohio State University ("University"), an instrumentality of the State of
Ohio, on the terms and conditions set forth hereinbelow, to cover commercial
development by OAI of Scanning Laser Imaging ("SLI") technology developed by Dr.
L. Xxxxx Xxxxx and co-workers at the University.
Definitions.
1.0. The following definitions shall apply throughout this document and any
correspondence between the parties relating to the subject matter thereof,
except insofar as the context clearly indicates a different meaning.
1.1. "Licensed Patent" means a patent or patent application identified in
Schedule A, and any other patent or patent application, including any division,
continuation, continuation-in-part, reissue or foreign counterpart, insofar as
it derives therefrom or covers an SLI invention developed by Xx. Xxxxx and
coworkers at the University prior to the effective date of this License
Agreement.
1.2. "Licensed Product" means (i) an SLI instrument or device the manufacture,
import, sale or use of which is covered by a Licensed Patent or (ii) a component
intended for use as part of or in connection with such instrument or device
insofar as the manufacture, import, sale or use of such component is covered by
a Licensed Patent.
1.3. "Licensed Service" means the use of Licensed Product and/or of a method
covered by a Licensed Patent.
License Scope.
2.0. OSURF hereby grants to OAI, and OAI hereby accepts, a worldwide license
under all Licensed Patents to manufacture, import, export, use, sell and lease
Licensed Products and Licensed Services, and to sublicense others to do so,
subject to the reservation of the right of the University to operate under
Licensed Patents for research and/or educational purposes, and to the other
provisions of this License Agreement.
2.1. This License is co-exclusive, by which is meant that OSURF may grant only
one additional license under Licensed Patents for Licensed Products or Licensed
Services for any
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combination of field use, geographical area and period of time for which this
License is in effect. A single additional license has been granted for all
fields, all geographical areas and the life of the licensed patents. If such
additional license shall terminate, then OAI shall be notified in writing and
shall have the option, for sixty (60) days following such notice, of converting
its co-exclusive License to exclusive, provided that this License (to OAI)
remains in effect, that OAI is not in default or breach of this Agreement and
that OAI agrees in writing to thereafter pay minimum royalties equal to twice
those specified in Section 4.4 hereof.
2.2. This License shall take effect with respect to any Licensed Patent upon the
later of (i) the effective date of this License Agreement and (ii) the filing of
a patent application which falls within the definition of Licensed Patent. This
License shall terminate with respect to any Licensed Patent upon the earlier of
(iii) the termination of this License Agreement and (iv) the expiration,
cancellation or final unappealable determination of invalidity of such Licensed
Patent or the abandonment of a patent application which falls within the
definition of Licensed Patent.
Licensee Performance.
3.0. OAI will diligently exert its reasonable best efforts to develop and
market, either directly or through sub-licensees, Licensed Products and/or
Licensed Services for a broad range of applications. In particular, OAI
undertakes to achieve, directly or through sub-licensees, the milestones of
sections 3.1-3.4. OAI will notify OSURF of its achievement, partial achievement
or lack of achievement of these milestones in a timely manner, and in the
absence of such notice, it will be presumed that the milestones have not been
achieved. These milestones are established to manifest and exemplify the
understanding of the parties and the commitment of OAI that continuous progress
will be made toward accomplishing this objective. It is recognized that specific
times or periods set forth in sections 3.1-3.4 may become unachievable due to
unanticipated and/or uncontrollable circumstances, and in such cases consent to
reasonable adjustment thereof, not to exceed doubling of any stated or implied
period, shall not be unreasonably withheld by OSURF. However, it is also
understood that such times or periods have been specified with reasonable
allowance for problems and delays that are typical of such projects, and in
particular only where funds substantially in excess of those reasonably
anticipatable prove to be required for the achievement of any milestone will be
the period for achieving such milestone be adjusted on the basis of shortage of
funds.
3.1. Not later than twelve (12) months after the effective date of this
Agreement, OAI will have assembled a prototype SLI device for demonstration
purposes.
3.2. Not later than thirty-six (36) months after the effective date of this
Agreement, OAI will have accumulated data suitable for submission to the FDA for
regulatory approval of a Licensed Product or Service.
3.3. Not later than thirty-six (36) months after the effective date of this
Agreement, OAI will submit to the FDA an application for regulatory approval of
a Licensed Product or Service.
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3.4. Not later than twenty-four (24) months after the effective date of this
Agreement OAI will have a Licensed Product on the market, it being the
contemplation of the parties that this milestone will be met by a Licensed
Product not requiring FDA approval.
3.5. Thereafter introduction of Licensed Products and/or Services for sale or
lease will, as rapidly as practicable, be actively promoted by OAI and/or
sub-licensees for a broad range of applications.
3.6. OAI and its sub-licensees will xxxx all Licensed Products subject to this
License Agreement with the numbers of all applicable U.S. Licensed Patents.
Royalties and Other Payments.
4.0. [Reserved]
4.1. OAI will pay royalties to OSURF based upon the net sales and leases by OAI
and sub-licensees of Licensed Products and Licensed Services. For such
transactions not at arms-length, the royalties will be based upon similar
transactions that are at arms-length. No royalties will be due OSURF upon resale
of a Licensed Product for which royalties have accrued in favor of OSURF on the
initial sale pursuant to 4.1-4.3 and 4.5 hereof. No royalties will be due OSURF
in respect of Licensed Services provided through the use of a Licensed Product
upon which royalties have been paid to OSURF, unless royalties are payable to or
at the direction of OAI or a sub-licensee in respect of such Licensed Service
(herein referred to as "Section 4.1 Service Royalties").
4.2. Subject to section 4.1 above, royalties due OSURF hereunder will be five
percent (5.0%) of cumulative net sales and leases of Licensed Products and
Licensed Services under authority of this License Agreement, except that
royalties will be payable to OSURF in the amount of twenty percent (20%) of
Section 4.1 Service Royalties accrued on or before the second anniversary of the
effective date of this Agreement and thirty-three and one-third percent
(33-1/3%) of Section 4.1 Service Royalties accrued thereafter.
4.3. Royalties accrued in favor of OSURF hereunder during each calendar quarter
will be paid to OSURF by OAI within forty-five (45) days after the end of such
quarter, accompanied by a concise statement of the basis upon which the amount
due was determined.
4.4. For complete calendar quarters following the third anniversary of the
effective date of this License Agreement, royalties of not less than the
following amounts will be deemed to have accrued hereunder in favor of OSURF:
Year 4 $ 5,000.00 per quarter
Year 5 7,500.00 per quarter
Year 6 10,000.00 per quarter
Year 7 15,000.00 per quarter
Year 8 20,000.00 per quarter
Thereafter 25,000.00 per quarter
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4.5. The fair value of any payment, thing, right or forbearance transferred to
or at the direction of OAI or a sub-licensee in exchange for the sale or lease
of Licensed Product or Licensed Service hereunder shall be included in the net
sale or lease amount upon which any royalties due OSURF hereunder are
calculated.
Records.
5.0. OAI and sub-licensees shall maintain records adequate to determine the
amount of royalties due to OSURF hereunder, and shall preserve such records and
make them available to OSURF for reasonable audit for three years following the
end of the period to which they pertain, or for such longer time as may be
necessary to finally resolve any questions that may be raised by OSURF. If any
audit reveals that payment of royalties due OSURF hereunder is delinquent by
$10,000.00 or more, OAI shall reimburse OSURF for its expenses in determining
and collecting the delinquent amount.
Term and Termination.
6.0. This License Agreement shall become effective upon execution by the last of
the parties to execute it.
6.1. Upon termination of this License Agreement all accrued royalties will
become due and payable within forty-five (45) days thereafter, and such
obligation, together with any other specific obligations of payment or otherwise
that have then matured, shall survive until discharged.
6.2. OAI may terminate this License Agreement at will upon written notice to
OSURF.
6.3. OSURF may terminate this License Agreement for breach of any material
provision thereof by OAI, including any of the undertakings set forth in
sections 3.0-3.6, particularly including the milestones set forth in sections
3.1-3.4, and in sections 4.1-4.5 and 8.2. OSURF shall notify OAI in writing of
any alleged breach of a material provision of this Agreement. OAI shall have
thirty (30) days after said notice to cure the alleged breach, after which
OSURF, subject to the terms of this Agreement, may terminate this License
Agreement.
6.4. Either party may terminate this License Agreement upon the insolvency or
bankruptcy of, or the seeking of protection from creditors by, the other.
6.5. Waiver of any breach shall not be deemed to impair the right of a party to
seek and obtain any remedy for any other breach.
Limitation of Liability.
7.0. OSURF makes no representations or warranties as to the accuracy or
reliability of any results or information provided in connection with this
License Agreement, or as to the validity or enforceability of any patent or
other rights conveyed hereunder, or as to the freedom of any Licensed Product or
Licensed Service hereunder from claim of infringement by others. IN
41
PARTICULAR, OSURF MAKES NO WARRANTIES OF MERCHANTABILITY OR OF FITNESS FOR ANY
PARTICULAR PURPOSE IN RESPECT OF ANY GOODS, SERVICES OR INFORMATION.
Furthermore, OSURF will not be liable for any consequential or punitive damages
in connection with the subject matter of this License Agreement, nor in any
event will its liability exceed the amounts it has received pursuant to this
License Agreement.
Miscellaneous.
8.0. This License Agreement shall be construed in accordance with the laws of
the State of Ohio, except for provisions relating to conflict of law.
8.1. Neither party shall use the name of the other, or of any affiliate or
sub-licensee of the other, in connection with any commercial promotion, except
with prior express written consent.
8.2. OAI acknowledges the position of OSURF and The Ohio State University that
the existing SLI prototype at the University will not be available for
commercial demonstrations and that, except with the express and fully informed
approval of the University's Associate Vice President for Research and its
Director of the Comprehensive Cancer Center, or their authorized delegates, (i)
no research, development and/or testing activities relating to SLI technology
and supported or arranged for by or on behalf of OAI or any other licensee or
sublicensee of the SLI technology will be undertaken by or under the auspices of
OSURF or the University, and any approved exceptions should not discriminate to
the unfair disadvantage of any other licensee, (ii) Xx. Xxxxx will not
participate in research or development activities conducted by or under the
auspices of OSURF or the University to develop the SLI technology, and (iii) Xx.
Xxxxx will neither directly nor beneficially own, control or have an option or
other claim to more than a five percent (5%) interest in either the equity or
the debt of OAI or its successors, assigns or sublicensees and whatever direct
or beneficial interest he has will be held in a voting trust over which he has
no direct or indirect control. OAI will not take, or authorize or encourage
others to take, any action to subvert such positions, will not accept or retain
the fruits of any subversion of them, and will promptly report to said Associate
Vice President for Research any evidence of which it becomes aware that others
may be attempting to subvert them. However, it shall not be deemed a breach of
the provisions of this section 8.2 for OAI to solicit informed approval of
exceptions to these positions.
8.3. This License Agreement incorporates by reference the Confidentiality
Agreement between the parties dated December 17, 1990, and with that
incorporation sets forth the entire understanding between the parties relating
to its subject matter, integrating all prior representations and negotiations.
SCHEDULE A
U.S. Patent No. (or Issue (or
Appl'n. No.) Filing Date) Name Inventor(s) Title
42
4,601,537 7/22/86 Xxxx X. Xxxxxxxx APPARATUS AND METHOD FOR FORMING IMAGES
AND FOR OPTICAL DEMULTI-PLEXING
4,758,727 7/19/88 L. Xxxxx Xxxxx METHOD AND APPARATUS FOR THE
Cornhill, F. MEASUREMENT OF LOW-LEVEL LASER-INDUCED
Xxxxxxxxx, J. FLUORESCENCE
Boninger, M.
4,877,966 10/31/89 L. Xxxxx Xxxxx METHOD AND APPARATUS FOR THE
Cornhill, F. MEASUREMENT OF LOW-LEVEL LASER-INDUCED
Xxxxxxxxx, J. FLUORESCENCE
Boninger, M.
5,037,207 08/06/91 L. Xxxxx Xxxxx IMPROVED LASER IMAGING SYSTEM
Xxxxxxxxx, J.
Cornhill, X.
Xxxx, Inching
(SN650,455) (02/04/91) L. Xxxxx Xxxxx IMPROVED LASER IMAGING SYSTEM
Xxxxxxxxx, J.
Cornhill, X.
Xxxx, Inching
AGREED TO AND ACCEPTED:
Optical Analytic, Inc.
By:_______________________________________________________Date__________________
Print name/title________________________________________________________________
The Ohio State University Research Foundation
By: /s/ Xxxxxx X. Xxxxxxx Date 8/16/91
--------------------------------
Print name/title Executive Director, Xxxxxx X. Xxxxxxx
The Ohio State University
By: /s/ X.X. Xxxxx Date August 16, 1991
--------------------------------
Print name/title Xxxxxx X. Xxxxx, Vice President for Research
43
Exhibit B2 to Xxxxxx-Xxxxx License Agreement
44
OPTICAL
ANALYTIC, INC.
------------------------------------------------------------------------
Xxxxx Xxxxx Xxxxxx Xxxx
0000 Xxxxxxxxx Xxxxx
Xxxxxx, Xxxx 00000
(000) 000-0000
November 23, 1993
VIA FACSIMILE
(000) 000-0000
Xx. Xxxxx Xxxxxx
President and Chief
Executive Officer
LXR Biotechnology, Inc.
0000 Xxxxxxxx Xxxxxxx
Xxxxxxxx, Xxxxxxxxxx 00000-0000
Dear Xxxxx:
Please find with this fax a copy of our signed License Agreement from Ohio State
and letter dated May 18, 1993, which extended the milestones to November 30,
1995, and removed the ownership caps on David's interest in the company.
It is my understanding that Ohio State will issue another extension letter
tomorrow. We will forward that letter along with the revised contracts at that
time.
Sincerely,
/s/ F. Xxxxx Xxxxx/bb
---------------------------
F. Xxxxx Xxxxx
FDR:bb
Enclosure
45
oaisli.lc4
LICENSE AGREEMENT
This License Agreement is entered into between Optical Analytic, Inc., ("OAI"),
a corporation of Ohio having a principal place of business at 0000 Xxxxxxxxx
Xx., Xxxxxx, Xxxx 00000, and The Ohio State University Research Foundation
("OSURF"), a non-profit corporation of Ohio having a principal place of business
at 0000 Xxxxx Xxxx, Xxxxxxxx, Xxxx 00000-0000 and acting on behalf of itself and
The Ohio State University ("University"), an instrumentality of the State of
Ohio, on the terms and conditions set forth hereinbelow, to cover commercial
development by OAI of Scanning Laser Imaging ("SLI") technology developed by Dr.
L. Xxxxx Xxxxx and co-workers at the University.
Definitions.
1.0. The following definitions shall apply throughout this document and any
correspondence between the parties relating to the subject matter thereof,
except insofar as the context clearly indicates a different meaning.
1.1. "Licensed Patent" means a patent or patent application identified in
Schedule A, and any other patent or patent application, including any division,
continuation, continuation-in-part, reissue or foreign counterpart, insofar as
it derives therefrom or covers an SLI invention developed by Xx. Xxxxx and
coworkers at the University prior to the effective date of this License
Agreement.
1.2. "Licensed Product" means (i) an SLI instrument or device the manufacture,
import, sale or use of which is covered by a Licensed Patent or (ii) a component
intended for use as part of or in connection with such instrument or device
insofar as the manufacture, import, sale or use of such component is covered by
a Licensed Patent.
1.3. "Licensed Service" means the use of Licensed Product and/or of a method
covered by a Licensed Patent.
License Scope.
2.0. OSURF hereby grants to OAI, and OAI hereby accepts, a worldwide license
under all Licensed Patents to manufacture, import, export, use, sell and lease
Licensed Products and Licensed Services, and to sublicense others to do so,
subject to the reservation of the right of the University to operate under
Licensed Patents for research and/or educational purposes, and to the other
provisions of this License Agreement.
2.1. This License is co-exclusive, by which is meant that OSURF may grant only
one additional license under Licensed Patents for Licensed Products or Licensed
Services for any
46
combination of field use, geographical area and period of time for which this
License is in effect. A single additional license has been granted for all
fields, all geographical areas and the life of the licensed patents. If such
additional license shall terminate, then OAI shall be notified in writing and
shall have the option, for sixty (60) days following such notice, of converting
its co-exclusive License to exclusive, provided that this License (to OAI)
remains in effect, that OAI is not in default or breach of this Agreement and
that OAI agrees in writing to thereafter pay minimum royalties equal to twice
those specified in Section 4.4 hereof.
2.2. This License shall take effect with respect to any Licensed Patent upon the
later of (i) the effective date of this License Agreement and (ii) the filing of
a patent application which falls within the definition of Licensed Patent. This
License shall terminate with respect to any Licensed Patent upon the earlier of
(iii) the termination of this License Agreement and (iv) the expiration,
cancellation or final unappealable determination of invalidity of such Licensed
Patent or the abandonment of a patent application which falls within the
definition of Licensed Patent.
Licensee Performance.
3.0. OAI will diligently exert its reasonable best efforts to develop and
market, either directly or through sub-licensees, Licensed Products and/or
Licensed Services for a broad range of applications. In particular, OAI
undertakes to achieve, directly or through sub-licensees, the milestones of
sections 3.1-3.4. OAI will notify OSURF of its achievement, partial achievement
or lack of achievement of these milestones in a timely manner, and in the
absence of such notice, it will be presumed that the milestones have not been
achieved. These milestones are established to manifest and exemplify the
understanding of the parties and the commitment of OAI that continuous progress
will be made toward accomplishing this objective. It is recognized that specific
times or periods set forth in sections 3.1-3.4 may become unachievable due to
unanticipated and/or uncontrollable circumstances, and in such cases consent to
reasonable adjustment thereof, not to exceed doubling of any stated or implied
period, shall not be unreasonably withheld by OSURF. However, it is also
understood that such times or periods have been specified with reasonable
allowance for problems and delays that are typical of such projects, and in
particular only where funds substantially in excess of those reasonably
anticipatable prove to be required for the achievement of any milestone will be
the period for achieving such milestone be adjusted on the basis of shortage of
funds.
3.1. Not later than twelve (12) months after the effective date of this
Agreement, OAI will have assembled a prototype SLI device for demonstration
purposes.
3.2. Not later than thirty-six (36) months after the effective date of this
Agreement, OAI will have accumulated data suitable for submission to the FDA for
regulatory approval of a Licensed Product or Service.
3.3. Not later than thirty-six (36) months after the effective date of this
Agreement, OAI will submit to the FDA an application for regulatory approval of
a Licensed Product or Service.
47
3.4. Not later than twenty-four (24) months after the effective date of this
Agreement OAI will have a Licensed Product on the market, it being the
contemplation of the parties that this milestone will be met by a Licensed
Product not requiring FDA approval.
3.5. Thereafter introduction of Licensed Products and/or Services for sale or
lease will, as rapidly as practicable, be actively promoted by OAI and/or
sub-licensees for a broad range of applications.
3.6. OAI and its sub-licensees will xxxx all Licensed Products subject to this
License Agreement with the numbers of all applicable U.S. Licensed Patents.
Royalties and Other Payments.
4.0. [Reserved]
4.1. OAI will pay royalties to OSURF based upon the net sales and leases by OAI
and sub-licensees of Licensed Products and Licensed Services. For such
transactions not at arms-length, the royalties will be based upon similar
transactions that are at arms-length. No royalties will be due OSURF upon resale
of a Licensed Product for which royalties have accrued in favor of OSURF on the
initial sale pursuant to 4.1-4.3 and 4.5 hereof. No royalties will be due OSURF
in respect of Licensed Services provided through the use of a Licensed Product
upon which royalties have been paid to OSURF, unless royalties are payable to or
at the direction of OAI or a sub-licensee in respect of such Licensed Service
(herein referred to as "Section 4.1 Service Royalties").
4.2. Subject to section 4.1 above, royalties due OSURF hereunder will be five
percent (5.0%) of cumulative net sales and leases of Licensed Products and
Licensed Services under authority of this License Agreement, except that
royalties will be payable to OSURF in the amount of twenty percent (20%) of
Section 4.1 Service Royalties accrued on or before the second anniversary of the
effective date of this Agreement and thirty-three and one-third percent
(33-1/3%) of Section 4.1 Service Royalties accrued thereafter.
4.3. Royalties accrued in favor of OSURF hereunder during each calendar quarter
will be paid to OSURF by OAI within forty-five (45) days after the end of such
quarter, accompanied by a concise statement of the basis upon which the amount
due was determined.
4.4. For complete calendar quarters following the third anniversary of the
effective date of this License Agreement, royalties of not less than the
following amounts will be deemed to have accrued hereunder in favor of OSURF:
Year 4 $ 5,000.00 per quarter
Year 5 7,500.00 per quarter
Year 6 10,000.00 per quarter
Year 7 15,000.00 per quarter
Year 8 20,000.00 per quarter
Thereafter 25,000.00 per quarter
48
4.5. The fair value of any payment, thing, right or forbearance transferred to
or at the direction of OAI or a sub-licensee in exchange for the sale or lease
of Licensed Product or Licensed Service hereunder shall be included in the net
sale or lease amount upon which any royalties due OSURF hereunder are
calculated.
Records.
5.0. OAI and sub-licensees shall maintain records adequate to determine the
amount of royalties due to OSURF hereunder, and shall preserve such records and
make them available to OSURF for reasonable audit for three years following the
end of the period to which they pertain, or for such longer time as may be
necessary to finally resolve any questions that may be raised by OSURF. If any
audit reveals that payment of royalties due OSURF hereunder is delinquent by
$10,000.00 or more, OAI shall reimburse OSURF for its expenses in determining
and collecting the delinquent amount.
Term and Termination.
6.0. This License Agreement shall become effective upon execution by the last of
the parties to execute it.
6.1. Upon termination of this License Agreement all accrued royalties will
become due and payable within forty-five (45) days thereafter, and such
obligation, together with any other specific obligations of payment or otherwise
that have then matured, shall survive until discharged.
6.2. OAI may terminate this License Agreement at will upon written notice to
OSURF.
6.3. OSURF may terminate this License Agreement for breach of any material
provision thereof by OAI, including any of the undertakings set forth in
sections 3.0-3.6, particularly including the milestones set forth in sections
3.1-3.4, and in sections 4.1-4.5 and 8.2. OSURF shall notify OAI in writing of
any alleged breach of a material provision of this Agreement. OAI shall have
thirty (30) days after said notice to cure the alleged breach, after which
OSURF, subject to the terms of this Agreement, may terminate this License
Agreement.
6.4. Either party may terminate this License Agreement upon the insolvency or
bankruptcy of, or the seeking of protection from creditors by, the other.
6.5. Waiver of any breach shall not be deemed to impair the right of a party to
seek and obtain any remedy for any other breach.
Limitation of Liability.
7.0. OSURF makes no representations or warranties as to the accuracy or
reliability of any results or information provided in connection with this
License Agreement, or as to the validity or enforceability of any patent or
other rights conveyed hereunder, or as to the freedom of any Licensed Product or
Licensed Service hereunder from claim of infringement by others. IN
49
PARTICULAR, OSURF MAKES NO WARRANTIES OF MERCHANTABILITY OR OF FITNESS FOR ANY
PARTICULAR PURPOSE IN RESPECT OF ANY GOODS, SERVICES OR INFORMATION.
Furthermore, OSURF will not be liable for any consequential or punitive damages
in connection with the subject matter of this License Agreement, nor in any
event will its liability exceed the amounts it has received pursuant to this
License Agreement.
Miscellaneous.
8.0. This License Agreement shall be construed in accordance with the laws of
the State of Ohio, except for provisions relating to conflict of law.
8.1. Neither party shall use the name of the other, or of any affiliate or
sub-licensee of the other, in connection with any commercial promotion, except
with prior express written consent.
8.2. OAI acknowledges the position of OSURF and The Ohio State University that
the existing SLI prototype at the University will not be available for
commercial demonstrations and that, except with the express and fully informed
approval of the University's Associate Vice President for Research and its
Director of the Comprehensive Cancer Center, or their authorized delegates, (i)
no research, development and/or testing activities relating to SLI technology
and supported or arranged for by or on behalf of OAI or any other licensee or
sublicensee of the SLI technology will be undertaken by or under the auspices of
OSURF or the University, and any approved exceptions should not discriminate to
the unfair disadvantage of any other licensee, (ii) Xx. Xxxxx will not
participate in research or development activities conducted by or under the
auspices of OSURF or the University to develop the SLI technology, and (iii) Xx.
Xxxxx will neither directly nor beneficially own, control or have an option or
other claim to more than a five percent (5%) interest in either the equity or
the debt of OAI or its successors, assigns or sublicensees and whatever direct
or beneficial interest he has will be held in a voting trust over which he has
no direct or indirect control. OAI will not take, or authorize or encourage
others to take, any action to subvert such positions, will not accept or retain
the fruits of any subversion of them, and will promptly report to said Associate
Vice President for Research any evidence of which it becomes aware that others
may be attempting to subvert them. However, it shall not be deemed a breach of
the provisions of this section 8.2 for OAI to solicit informed approval of
exceptions to these positions.
8.3. This License Agreement incorporates by reference the Confidentiality
Agreement between the parties dated December 17, 1990, and with that
incorporation sets forth the entire understanding between the parties relating
to its subject matter, integrating all prior representations and negotiations.
SCHEDULE A
U.S. Patent No. (or Issue (or
Appl'n. No.) Filing Date) Name Inventor(s) Title
50
4,601,537 7/22/86 Xxxx X. Xxxxxxxx APPARATUS AND METHOD FOR FORMING IMAGES
AND FOR OPTICAL DEMULTI-PLEXING
4,758,727 7/19/88 L. Xxxxx Xxxxx METHOD AND APPARATUS FOR THE
Cornhill, F. MEASUREMENT OF LOW-LEVEL LASER-INDUCED
Xxxxxxxxx, J. FLUORESCENCE
Boninger, M.
4,877,966 10/31/89 L. Xxxxx Xxxxx METHOD AND APPARATUS FOR THE
Cornhill, F. MEASUREMENT OF LOW-LEVEL LASER-INDUCED
Xxxxxxxxx, J. FLUORESCENCE
Boninger, M.
5,037,207 08/06/91 L. Xxxxx Xxxxx IMPROVED LASER IMAGING SYSTEM
Xxxxxxxxx, J.
Cornhill, X.
Xxxx, Inching
(SN650,455) (02/04/91) L. Xxxxx Xxxxx IMPROVED LASER IMAGING SYSTEM
Xxxxxxxxx, J.
Cornhill, X.
Xxxx, Inching
51
AGREED TO AND ACCEPTED:
Optical Analytic, Inc.
By: /s/ Xxxxx XxXxxxxxx Date 8/15/91
--------------------------------
Print name/title Xxxxx XxXxxxxxx, President
The Ohio State University Research Foundation
By: /s/ Xxxxxx X. Xxxxxxx Date 8/16/91
--------------------------------
Print name/title Xxxxxx X. Xxxxxxx/Executive Director
The Ohio State University
By: /s/ Xxxxxx X. Xxxxx Date August 16, 1991
--------------------------------
Print name/title Xxxxxx X. Xxxxx, Vice President for Research
52
----------------------
T . H . E Office of Technology Transfer 0000 Xxxxx Xxxx
XXXX Xxxxxxxx, XX 00000-0000
STATE
UNIVERSITY May 18, 1993 Phone 000-000-0000
FAX 000-000-0000
----------------------
Xxxxx X. XxXxxxxxx
Optical Analytic, Inc.
0000 Xxxxxxxxx Xxxxx
Xxxxxx, XX 00000
Re: Amendment of License Agreement of August 26, 1991 for SLI
Technology (Original Document and Identified by Running Header
"oaisli.1c4")
Dear Xx. XxXxxxxxx:
In response to your requests, and in view of Optical Analytical's efforts to
date to press forward after the termination of your joint venture with
colicensee Xxxxxx, Inc. and of OAI's plans for the future, we are willing to--
(i) consent to adjusting the milestone deadlines specified in
sections 3.1 through 3.4,
(ii) waive non-compliance by OAI with the milestone deadline
specified in section 3.1 (as extended by letter dated August 12, 1992), and
(iii) release OAI from the restrictions on Xx. Xxxxx'x
interest in OAI and related entities specified in section 8.2 (iii) --
by amending the above-identified License Agreement as follows:
A. In each of the sections 3.1. through 3.4., inclusive, the period for
meeting their respective milestone is extended by fifteen (15) months
beyond that specified in the original Agreement, and any default by OAI
prior to the effective date of this amendment in meeting the original
deadline for the milestone in any of these sections is waived;
provided, however, that the SLI devices referred to, directly or by
implication, in these sections are based on second-generation
multiparametric SLI technology.
B. In section 8.2., the test reading --
and (iii) Xx. Xxxxx will neither directly nor beneficially
own, control or have an option or other claim to more than a
five percent (5%) interest in either the equity or the debt of
OAI or its successors, assigns or sublicensees and whatever
direct or beneficial interest he has will be held in a voting
trust over which he has no direct or indirect control--
is deleted.
53
C. These amendments will be effective as of the date when this document
has been executed below by all parties.
If these amendments are acceptable, please have all three copies signed below
where indicated and return two copies to me.
Sincerely,
/s/ Xxxxx X. Xxxxxxx
---------------------------
Xxxxx X. Wildens
AGREED AND ACCEPTED--
The Ohio State University Research Foundation
By: /s/ X.X. Xxxxxxx Date: 5/19/93
--------------------------------
Print name/title: Xxxxxx X. Xxxxxxx, Executive Director
The Ohio State University
By: /s/ Xxxxx X. Xxxxxxxxx Date: 18 May 93
--------------------------------
Print name/title: Xxxxx X. Xxxxxxxx, Vice President
By: /s/ Xxxxx X. XxXxxxxxx Date: 5/21/93
--------------------------------
Print name/title: Xxxxx X. XxXxxxxxx -President
54
----------------------
T . H . E Office of Technology Transfer 0000 Xxxxx Xxxx
XXXX Xxxxxxxx, XX 00000-0000
STATE
UNIVERSITY August 31, 1993 Phone 000-000-0000
FAX 000-000-0000
----------------------
Xxxxx X. XxXxxxxxx
Optical Analytic, Inc.
0000 Xxxxxxxxx Xxxxx
Xxxxxx, Xxxx 00000
Subject: Option Under License Agreement of August 26, 1991 for SLI Technology
(Original Document Identified by Running Header "oaisli.lc4"), as
Amended by a Document Dated May 18, and Effective May 21, 1993.
Dear Xx. XxXxxxxxx:
Pursuant to Section 2.1 of the above-identified license agreement, we hereby
inform you that we have given notice to the additional (co-exclusive) licensee
terminating that additional (co-exclusive) license for cause, effective (as
permitted by the terms of that license) upon receipt of our notice, which the
carrier's proof of delivery return shows was no later than July 30, 1993. We
have had no response to that notice (or to an earlier notice dated July 7, 1993
for which the carrier has been unable to produce proof of delivery). In any
case, we regard that additional (co-exclusive) license as terminated, and that
option provisions of Section 2.1 as in effect as of September 1, 1993.
If you wish to exercise that option, as I understand from Xxxx Xxxx you do, you
may do so according to the EXERCISE OF OPTION language below by signing where
indicated on all three original counterparts of this letter and returning them
to me within sixty days of this notice for affixation of our signatures,
whereupon one will be returned to you. Please let me know if you have any
questions.
Sincerely,
/s/ Xxxxx X. Xxxxxxx
-----------------------
Xxxxx X. Xxxxxxx
EXERCISE OF OPTION
Optical Analytic, Inc., having received the foregoing notice of termination of
the additional (co-exclusive) license referred to in Section 2.1 of the
above-identified license agreement, hereby
55
exercises its option under such Section 2.1 to convert its license to an
exclusive license and, in accordance with the provisions of such Section 2.1,
will hereafter pay minimum royalties equal to twice those specified in Section
4.4 of such above-identified license agreement.
Optical Analytic, Inc.
By /s/ Xxxxx X. XxXxxxxxx Date September 7, 1993
----------------------------
Print name/title Xxxxx X. XxXxxxxxx, President
The Ohio State University Research Foundation and The Ohio State University
accept the above EXERCISE OF OPTION and recognize the above-identified license
agreement as amended in accordance therewith as of the date written adjacent the
signature above.
The Ohio State University Research Foundation
By: /s/ X.X. Xxxxxxx Date 9/23/93
----------------------------
The Ohio State University
By /s/ Xxxxx X. Xxxxxxxx Date 17 Sep 93
----------------------------
Xxxxx X. Xxxxxxxx, V.P. Business & Admin.
56
Exhibit B3 to Xxxxxx-Xxxxx License Agreement
57
----------------------
T . H . E Office of Technology Transfer 0000 Xxxxx Xxxx
XXXX Xxxxxxxx, XX 00000-0000
STATE
UNIVERSITY August 31, 1993 Phone 000-000-0000
FAX 000-000-0000
----------------------
Xxxxx X. XxXxxxxxx
Optical Analytic, Inc.
0000 Xxxxxxxxx Xxxxx
Xxxxxx Xxxx
Subject: Amendment of License Agreement of August 26, 1991 for Scanning Laser
Imaging Technology and Amendment to said License in Xxxxxxx to
XxXxxxxxx letter dated May 28,1993.
Dear Xx. XxXxxxxxx:
In response to your requests and in view of your further efforts to
commercialize the Scanning Laser Imaging Technology (SLI) developed at
UNIVERSITY (referring hereinafter collectively to The Ohio State University and
The Ohio State University Research Foundation), and with the realization that
Optical Analytic, Inc. (OAI) will have the exclusive license to develop and
commercialize this technology, the University is willing to:
a. extend the milestone deadlines specified in sections 3.1 through 3.4
beyond the maximum contemplated in Section 3.0, and extend the period
till the minimum royalties are required.
b. provide restricted permission for the use of UNIVERSITY's name in
documents describing the relationship between UNIVERSITY and OAI with
respect to the SLI,
in return for OAI's agreement to reimburse certain patenting expenses.
To achieve these ends the subject License Agreement is amended as follows:
1. In each of the sections 3.1. through 3.4. inclusive, the period for
meeting the respective milestone is extended by twenty-four (24) months
beyond that specified in the original agreement and any default by OAI
prior to the effective date of this amendment in meeting the original
deadline for the milestone in any of these sections is waived; provided
however, that the SLI devices referred to directly or by implication,
in these sections are based on second-generation multiparametric SLI.
2. Amend section 4.4. to state that minimum royalties begin after the
fourth anniversary of the original agreement and that those minimums
are $5,000., $7,500., $10,000., $15,000., $20,000. and $25,000. per
quarter for Year 5, Year 6, Year 7, Year 8, Year 9, and Thereafter,
respectively.
58
3. Insert section 4.0.:
OAI shall reimburse UNIVERSITY for patenting expenses, for the patents
which are the subject of this license agreement, billed after the date
of this letter. Reimbursement shall be within sixty (60) days of
receipt by OAI of an invoice from UNIVERSITY.
Section 8.1 restricts the use by either party of the name of the other party for
use in any commercial promotion. Permission is hereby given for use of the name
in a legal Prospectus only to refer to UNIVERSITY for the limited purpose of
giving the information that the SLI was invented at UNIVERSITY and that OAI has
an exclusive license from UNIVERSITY to commercialize the SLI. No other use is
permitted without prior written permission from UNIVERSITY.
These amendments will be effective as of the date when this document is executed
below by all parties.
Regards,
Xxxx X. Lafayatis
Director, Technology Transfer
AGREED AND ACCEPTED:
The Ohio State University Research Foundation
By: /s/ X.X. Xxxxxxx Date: 11/29/93
----------------------
Title: Executive Director
The Ohio State University
By: /s/ X.X. Xxxxxxx Date: 11/29/93
----------------------
Title: Assoc VP - Research
Optical Analytic, Inc.
By: /s/ Xxxxx X. XxXxxxxxx Date: 11/29/93
----------------------
Title: President
59
Exhibit B4 to Xxxxxx-Xxxxx License Agreement
60
AMENDMENT TO SLDI LICENSE AGREEMENT
The License Agreement among Optical Analytic, Inc., The Ohio State Research
Foundation and The Ohio State University dated August 26, 1991, as amended to
date ( the "License"), is hereby further amended to delete paragraphs 3.2 and
3.3 of the License, effective as of July 10, 1996.
OPTICAL ANALYTIC, INC.
By: /s/ L. Xxxxx Xxxxx
----------------------------
L. Xxxxx Xxxxx, President
THE OHIO STATE UNIVERSITY
By: /s/ Xxxx X. Lafayatis
----------------------------
Its: Director, Technology Transfer
THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION
By: /s/ Xxxx X. Lafayatis
----------------------------
Its: Director, Technology Transfer
61
Exhibit B5 to Xxxxxx-Xxxxx License Agreement
62
AMENDMENT TO SLDI LICENSE AGREEMENT
The License Agreement among Optical Analytic, Inc. ("OAI"), The Ohio
State Research Foundation ("OSRF") and The Ohio State University ("OSU") dated
August 26, 1991, as amended to date ( the "License"), is hereby further amended
to delete U.S. Patent No. 4,601,537, titled "APPARATUS AND METHOD FOR FORMING
IMAGES AND FOR OPTICAL DEMULTIPLEXING" (the "'537 Patent"), from Schedule A to
the License, and thereby from the definition of "Licensed Patent" in Section 1.1
of the License, so that the '537 Patent is no longer licensed to OAI under the
License.
All other provisions of the License shall remain in full force and
effect.
IN WITNESS WHEREOF, the parties hereto have entered into this Amendment
as of August 27, 1996.
OPTICAL ANALYTIC, INC.
By:
----------------------------
L. Xxxxx Xxxxx, President
THE OHIO STATE UNIVERSITY
By:
----------------------------
Its: Vice President for Business & Admin.
THE OHIO STATE UNIVERSITY RESEARCH FOUNDATION
By:
----------------------------
Its: Director, Technology Transfer
63
Exhibit C1 to Xxxxxx-Xxxxx License Agreement
64
US PATENT 5,037,207
<=2> GET 1st DRAWING SHEET OF 74
Aug. 6, 1991
Laser imaging system
INVENTOR: Tomei, L. Xxxxx, Columbus, Ohio
Xxxxxxxxx, Jogikal, Columbus, Ohio
Cornhill, Xxxx, Worthington, Ohio
Chen, Inching, Columbus, Ohio
ASSIGNEE-AT-ISSUE: Ohio State University Research Foundation, Columbus, Ohio
(02)
APPL-N0: 174,977
FILED: Mar. 28, 1988
REL-US-DATA:
Continuation-in-part of Ser. No. 828,651, Feb. 12, 1986 now abandoned
INT-CL: [5] X00X 0#00
XX-XX: 356#444; 250#458.1; 356#344; 356#417
CL: 356;250
SEARCH-FLD: 356#344, 444, 317, 318, 417; 250#458.1, 459.1, 461.1, 461.2
REF-CITED:
U.S. PATENT DOCUMENTS
3,450,455 6/1969 * Xxxxxx 356#444
3,516,746 6/1970 * Shibata et al. 356#319
3,981,590 9/1976 * Xxxxxxx 356#419
4,107,534 8/1978 * Piltingsrud 250#368
4,523,799 6/1985 * Delhaye 350#6.3
4,586,781 5/1986 * Xxxxxxx et al. 350#3.7
4,758,727 7/1988 * Tomei et al. 250#461.2
FOREIGN PATENT DOCUMENTS
65
0123942 11/1984 * European Patent Organization
0155813 9/1985 * European Patent Organization
OTHER PUBLICATIONS
Xxxxxx, F., "Mural Television Display Using Fiber Optics", RCA Technical Notes
(RCA TN No. 188, 1958).
Xxxx et al., "Vidicon Microscope for Counting Fluorescent Particles", Review of
Scientific Instruments, vol. 24, No. 4 (1971), p. 508.
Xxxxxx et al., "A Laser Flying Spot Scanner for Use in Automated Fluorescence
Antibody Instrumentation", Journal of the Association for the Advancement of
66
PAGE 2
Xxx. No. 5037207, *
Medical Instrumentation, vol. 6, No. 3, May-Jun. 1972.
Bussini et al., "A Silicon Rubber Scintillation Compound for Complex Geometry
Radiation Detectors", Nuclear Instruments and Methods, No. 2, (1973), pp.
333-335.
"The New FACS 400 Series, FACS 440, FACS 420, FACS 400, Fluorescence Activated
Cell Sorting", Becton Xxxxxxxxx FACS Systems, 1981.
"The FACS Analyzer Fluorescence and Volume Cell Analysis" Brochure, Becton
Xxxxxxxxx FACS Systems, 1982.
Young et al., IEEE Transactions on Biomed. Engineering, BME-29;2, 1983.
Xxxxxxxxx et al., IEEE Transactions on Biomed. Engineering, BME-33, 1984.
"Photodigitizing with PDS Microdensitometer Systems" Brochure, Xxxxxx-Xxxxx, not
dated.
"Microdensitometer 3 CS" Brochure, Xxxxx-Xxxxx, not dated.
"Microdensitometer 6" Brochure, Xxxxx-Xxxxx, not dated.
"EIKONIX Registered TM Applied Research Software and Product Development
Feasibility and Impact Studies Automated Instruments" Brochure, EIKONIX
Registered TM Corporation, not dated.
"ACAS 470 Workstation" Brochure, Meridian Instruments, Inc., not dated.
"Cytoscan-Cytogenetic Scanning Analyser System" Brochure, Image Recognition
Systems, Ltd., not dated.
PRIM-EXMR: Xxxxxxxxxxx, Xxxxxxx X.
LEGAL-REP: Emch, Schaffer, Xxxxxx & Xxxxxxxx Co.
ABST:
A laser imaging system is disclosed which provides the versatility of wide
field digital imaging with enhanced spatial resolution and light gathering
efficiency. The system will scan targets of any size, dependent only upon the
data retrieval and storage limitation of the computer support system, for
forward light loss densitometry images as well as fluorescent and forward
scatter images. The system is easily adaptable for rare event detection and
tracking. The laser system will provide image capture of an entire target within
10 to 60 seconds and controls the scan of the laser beam in three-dimensional
pattern and speed. The beam may be repositioned to any one of 16 million
locations on a target within an accuracy of + / - 0.5 um. Finally, the imaging
system of the present invention utilizes a novel optical fiber based detector
assembly having NA values of 0.58-0.95 and filters having less than 15% loss at
emission wavelengths. Thus, the imaging system of the present invention can
capture from 14% to 32% of total fluorescence emission.
67
NO-OF-CLAIMS: 11
EXMPL-CLAIM: <=10> 1
NO-OF-FIGURES: 77
NO-DRWNG-PP: 74
PARCASE:
This is a continuation-in-part of copending application Ser. No. 828,651
filed on Feb. 12, 1986, now abandoned.
68
PAGE 3
Xxx. No. 5037207, *
SUM:
BACKGROUND OF THE INVENTION
The laser imaging system of the present invention is designed to provide a
means for rapid quantitative image capture and digitization where requirements
for field size, speed, spatial resolution, dynamic range, and low light
sensitivity are not adequately provided for by conventional optical imaging
devices.
Specifically, optical devices that incorporate lenses to form a real or
virtual image at any point in the system are limited with respect to the target
field size, given a particular effective numerical aperture (NA) and spatial
resolution. For instance, a microscope provides a means of imaging using lenses
of high light gathering power, i.e., high numerical aperture (NA). Typically the
value of the NA for an objective lens with 20X is 0.50 (range 0.40-0.75).
However, this type of lens is capable of imaging a field of only 3 square
millimeters. See for example FIG. 1. If a field of 20 mm x 40 mm is to be imaged
(approximately the area of a standard microscope slide), then the maximum
effective NA for conventional optical lenses would be approximately 0.04-0.10.
For this reason highly sensitive fluorescence measuring devices have
incorporated microscopes with high NA lenses equipped with mechanical stages to
move the target and effect the wide field scan.
Gross mechanical translation of targets such as microscope slides is
generally slow and subject to maintenance problems related to wear and failure.
The ability of mechanical stages to physically move the target is limited by the
need for high positioning accuracy which is typically within 0.5 um. Thus, a
high precision autostage will have a maximal translation rate of only about 20
mm/second. Other additional complicating factors involved in image capture
require that the mechanical stage stop at each field location long enough for
the capture device to electronically transfer the image data to some storage
form. Thus, these conventional optical devices, when used for high resolution,
low light level scans (e.g., immunofluorescence), can require several hours to
capture target fields the size of a standard microscope slide.
If a field of 20 mm x 40 mm is to be imaged, then compromises are necessary
when using optical lenses. Wide field imaging with effective numerical apertures
of 0.9 or higher has led to complex designs and instruments that require hours
for image capture. A significant improvement upon these conventional optical
devices is the flying spot scanner. However, the flying spot scanners available
69
today including the most recent laser-induced devices, have a significant
problem, which is the simple fact that the focal point of the light beam is
fixed. Therefore, for a two dimensional scan, the beam spot is found on a curved
surface. Correction for this curvature in the present day flying spot scanner
requires the incorporation of cumbersome multisided spinning mirrors of complex
design. These mirrors afford little control over the location of the laser beam.
Conventional flying spot scanners also move the laser in a preprogrammed
ballistic direction. The direction and velocity parameters are preprogrammed and
the scan cannot be controlled outside of the programming.
Another problem associated with the design of a flying spot scanner for use
in fluorescence detection or forward scatter detection is the relatively low
efficiency light gathering available using conventional design which use lenses
for detection. For instance, a design described by Xxxxxx, et al., (J. Assoc.
70
PAGE 4
Xxx. No. 5037207, *
Adv. Medical Instr., 6:230, 1972) for a flying spot fluorescence detection is
capable of capturing less than 1% of the total target emission. This is
equivalent to an unacceptable NA of 0.10.
Imaging systems can be designed for a large variety of applications.
Generally, however, the requirements of target field size, NA, and spatial
resolution dictate specific structures for specific applications. There is
little crossover in design for the varying applications. The imaging system of
the present invention overcomes the design limitations of current imaging
devices and is easily adaptable to perform a large number of imaging operations.
SUMMARY OF THE INVENTION
The laser imaging system of the present invention eliminates the need for
mechanical translation stages for targets. The system is capable of scanning
targets of any size without gross stage movement subject only to the limitations
of available data processing and storage capabilities. The imaging provided by
the present invention is based not only upon optical density and forward light
loss (FLL) densitometry, but light scatter and fluorescence emission as well.
The scanning system can also be programmed to scan for rare event detection and
tracking, (i.e., the following of a neutron path in a target). The system will
provide image capture of an entire target within 10 to 60 seconds and controls
the scan of the laser beam in both pattern and speed. The beam spot may be
repositioned to any one of 16 million locations on a target within an accuracy
of + / - 0.5 um. Finally, the imaging system of the present invention utilizes a
novel optical fiber based detector assembly having NA values of 0.58-0.95 and
filters having less than 15% loss at emission wavelengths. Thus, the imaging
system of the present invention can capture from 14% to 32% of total
fluorescence emission.
The imaging system is easily adaptable to undertake a large variety of
imaging applications. The system will perform Clonogenic Assays and cDNA and
genomic DNA imaging. The system will perform multiwell plate assays such as a
fluorescence assay from any immobilized immunofluorescence assay system. Another
application is the imaging of submicron particles based on scatter
characteristics. Other applications include the discrimination of plane
polarized fluorescence emissions and qualitative and quantitative imaging of
those emissions. The imaging system of the present invention will perform
neuroautoradiographs yielding quantitative digital images in neutral density or
digital color. Yet another application is the migration inhibition assay using
multiwell plates for bone marrow transplantation testing. The imaging system of
the present invention will perform 3D Interferometry Surface Profiling for
71
non-contact micro-surface quantitative analysis. Another application for the
imaging system is the measurement of scatter or density for a cytochemistry
analysis of large sample screening.
The imaging system of the present invention performs opaque gel scanning as
well as transparent or semitransparent (i.e., translucent) gel scanning.
Scanning densitometry is used to quantitate typical translucent gels such as
agarose gels following chemical staining of the DNA, RNA, or protein bound to
the gel. However, the high degree of biochemical resolution required for gene
sequence analyses has resulted in the development of analytical techniques which
involve the electrophoretic transfer of material from translucent separation
gels to opaque membranes typically constructed of glass fibers or nylon matrix.
The material on these membranes is visualized by introducing a chemical stain to
colorimetrically xxxx the position and amount of the material. The material
72
PAGE 5
Xxx. No. 5037207, *
can also be labeled with a radioactive marker for subsequent analysis using
autoradiographic techniques and additional photosensitive elements such as x-ray
film. The membrane matrix prevents the use of conventional optical scanning
densitometry techniques because of the high light diffusion and attenuation
produced by the matrix. This is comparable to a high resolution image located on
a single side of heavy photographic paper. The optical fiber detector assembly
of the laser imaging system of the present invention collects all light, from
the entire scanned area, emitted at angles up to 90o from the optical axis
simultaneously at each and every position of the laser spot and assigns the
value of the light intensity to that individual position or pixel. Within
seconds, this process is performed several million times and a quantitative
digital reproduction is obtained of the image on the surface of the stained
membrane, photographic paper, or similar target scanned. Since this differs from
simple axial light loss or optical density, it is referred to as Forward Light
Loss (FLL) scanning.
Finally, the imaging system will perform the scanning of fluorescence
shadowing and forward scatter fluorescence shadowing through the use of a
fluorescent sensor, thus eliminating the problems of measuring the laser spot
size and focusing the spot.
These applications of the present invention are currently known, however,
this list is not intended to be limiting upon the range of potential uses for
this system.
DRWDESC:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the relationship of the numerical aperture (NA) and
target field image size for conventional lenses.
FIG. 2 is a schematic illustration of the laser imaging system of the present
invention.
FIG. 3 is an illustrative representation of a typical microscope slide and a
pattern of laser scanning the slide.
FIG. 4 is an illustrative representation of the light detection properties of
a blunt-cut optical fiber faceplate and a bias-cut optical fiber faceplate.
FIG. 5 is a schematic illustration of a typical detector assembly as used
73
with the present invention.
FIG. 6 is a schematic illustration of a detector assembly for use with the
present invention in detecting fluorescence and forward light scatter.
FIG. 7 is a conceptical illustration of the detector assembly of FIG. 6.
FIG. 8 is a graph showing optical fiber polarimetry for square
cross-sectional optical fibers.
FIG. 9 is an illustrative schematic of the plane polarization by a square
cross-sectional optical fiber of fluorescence emissions.
74
PAGE 6
Xxx. No. 5037207, *
FIG. 10A is a schematic block diagram of the image memory controller and
image memory array for the imaging system of the present invention.
FIG. 10B is a schematic block diagram of the CRT controller and graphics
memory array for the imaging system of the present invention.
FIG. 11 is a schematic illustration of the light acceptance cone of a
bias-cut optical fiber faceplate.
FIGS. 12A, B, C and D are schematics for the Image Memory Controller of the
present invention showing the circuitry for the Memory Request Arbiters and the
Input/Out Access circuitry.
FIGS. 13A, B, C and D are schematics for the Image Memory Controller of the
present invention showing the circuitry for the Memory and the Input/Output
Address Decoder.
FIGS. 14A, B, C and D are schematics for the Image Memory Controller of the
present invention showing the circuitry for the Memory Refresh Function and the
Interrupt Request Function and Data Transfer Acknowledgement Generation.
FIGS. 15A, B, C and D are schematics showing the Address Multiplexer
circuitry for the Image Memory Controller of the present invention.
FIGS. 16A and B are schematics showing the Memory Buffers circuitry for the
Image Memory Controller of the present invention.
FIGS. 17A and B are schematics showing the Input/Output registers circuitry
for the Image Memory Controller of the present invention.
FIGS. 18A, B, C and D through 20A, B, C and D are schematics showing the
Memory Chip Array circuitry for the Image Memory Array of the present invention.
FIGS. 21A, B, C and D are schematics showing the Memory Arrays, Address
liners, and Control Bus circuitry for the Image Memory Array of the present
invention.
FIGS. 22A, B, C and D are schematics showing the Image Data Shift Register
circuitry for the Image Memory Array of the present invention.
FIG. 23 is a schematic showing the Memory Array circuitry for the Image
75
Memory Array of the present invention.
FIGS. 24A, B, C and D are schematics showing the Video Display Generator
Interface circuitry for the CRT controller of the present invention.
FIGS. 25A, B, C and D are schematics showing the Address Decoder, Graphics
Memory Interface and Interrupt Request and Acknowledge circuitry of the CRT
Controller of the present invention.
FIGS. 26A, B, C and D are schematics showing the Zoom Control and Look-up
Table Circuitry for the CRT Controller of the present invention.
FIGS. 27A, B, C and D are schematics showing the Memory Chip Array circuitry
for the Graphics Memory Array of the present invention.
76
PAGE 7
Xxx. No. 5037207, *
FIGS. 28A, B, C and D are schematics showing the Graphics Display and Shift
Register circuitry for the Graphics Memory Array of the present invention.
FIGS. 29A, B, C and D are schematics showing the D/A Converter circuitry for
the Graphics Memory Array of the present invention.
DETDESC:
DESCRIPTION OF THE PREFERRED EMBODIMENT
The laser imaging system of the present invention provides wide field digital
imaging with enhanced spatial resolution and light gathering efficiency.
Referring now to FIG. 2, the optical design used with the imaging system is
shown. The primary laser 10, a 25 mW He-Cd, Omnichrome 450X, provides a beam 12
to a beam expander 14 composed of an objective lens and a 50 um spatial filter.
The beam 12 exits the beam expander 14 as an input collimated beam 16, 10 mm in
diameter. A three dimensional beam position controller 18, manufactured by
General Scanning Corp., receives the collimated beam 16.
The beam expander 14 ensures the utilization of the greatest possible surface
area of the mirrors of the beam controller 18. The beam 12 from the laser 10 is
too small and the beam expander 14 provides an adjustable spot size from 1 mm to
10 mm according to the needs of the controller 18. The beam expander 14 can be
modified optically to take advantage of Bessel Function beam characteristics if
desired. Such beam characteristics are evidenced by diminishing diffraction
rings which circle around the center spot. The optics of the beam expander 14
can be modified by replacement of the spatial filter with a circular disc to
block the high energy diffraction rings. Such a modification focuses the imaging
beam to a carefully selected spot size without any unwanted spillover.
The beam controller 18 includes an imaging lens (not shown) which produces a
2-10 um diameter spot depending on the diameter of the collimated beam 16.
Galvanometrically driven mirrors (not shown) incorporated within the beam
controller 18 provide for control over the 2-10 um diameter spot in the X, Y and
Z axes as the spot is focused on the target plane 20. The beam controller 18
allows for maximum scanning rates up to 100 Hz or an approximate spot vector
rate of 800 cm/sec. Each of the three axes of the beam controller 18 are under
individual software control which permits any desired scanning or tracking
pattern to be performed. Placement of the laser spot is accurate within + / -
0.5 um, thus the laser spot can be fixed on any specified cell in the target 20
77
area after scanning. Such placement accuracy permits further imaging at a higher
resolution and the recording of cell location for further direct examination if
desired.
The 3-D beam controller 18 manufactured by General Scanning, Inc.,
incorporates independent temperature control on all three main galvanometer
drives which maintains the temperature at 40 ( + / - 0.5) degrees C. The upper
scanner and lower laser xxxxxxxx incorporate sensors (not shown) for continuous
monitoring of ambient temperature during operation. The He-Cd or Argon-ION laser
in the lower chamber is provided with ducted positive ventilation using room air
in order to minimize environmental contamination and control the operating
temperatures of the optical components.
78
PAGE 8
Xxx. No. 5037207, *
As shown in FIG. 4, the imaging system can scan the entire sample area of a
microscope slide of prepared cells which will include 5 million to 20 million
cells. The system is driven at rates up to 100 Hz, which means that a single
line can be scanned in 0.005 seconds. The system can be programmed to perform
either vector scans or raster scans equally efficiently. The X and Y steps are
variable from 0.6 um to 40 mm in 0.6 um units. To illustrate the effectiveness
of this approach to scanning blood smears, the system is capable of detecting a
single positive fluorescent cell on a slide area of 400 sq. mm. (which can
include as many as 20 million cells), measure the fluorescence emission level
(up to 12 bit resolution), and specify the location of the cell within an
accuracy of + / - 0.5 um.
The basic detector assembly 22 is comprised of three basic elements: a high
numerical aperture optical fiber faceplate 24; a diffusion assembly 26 and a
photomultiplier tube (PMT) 32. The basic diffusion assembly 26 includes flashed
opal diffusion filters 30 and an internally reflective cylindrical coupling 28.
Two basic configurations of the optical fiber faceplate 24 are utilized
dependent on whether the imaging is to be based upon light transmission or
fluorescence emission within the target.
Referring to FIG. 4, laser imaging based upon detection of differences in
optical density [i.e., wide angle forward light loss (FLL)] utilizes a blunt-cut
high numerical aperture optical fiber faceplate 24 to collect and transmit light
to the PMT 28. However, for imaging fluorescence emissions the primary beam is
initially blocked on axis by introducing a bias end cut on the optical fibers.
Referring to FIG. 11, the acceptance cone of the component fibers is "tipped" by
angle B which is related to the bias end cut angle A as follows:
B = sin< - 1> [(N2/N1) sin A] - A
where N1 and N2 are the indices of refraction of air and the fiber core
respectively. The numerical aperture is not significantly changed when A is
approximately 30 degrees and, therefore, the light gathering power of the fibers
is not diminished when the incidence angle of the excitation beam remains close
to 90o however, the energy at the PMT is reduced more than 99%.
The PMT 32 is a high gain, low noise unit (Hamamatsu) having a 300-650 nm
response (max. 400 nm) typically operated at 600 to 1250 volts. It is critical
that transmitted light be diffused uniformly across the 2 inch diameter PMT 32
window to minimize variations in output related to spot position on target
plane. This is effectively accomplished by the combination of inherent annular
79
ray rotation at fiber output the optical and the diffusion assembly 26 which
includes the cylindrical internally reflective coupling 28.
Referring now to FIG. 5, the basic detector assembly 22 for standard
densitometric imaging of translucent objects by a laser beam is shown. The
detector assembly includes a blunt-cut optical fiber faceplate 24 and a
diffusion assembly 26 composed of a internal reflectance tube 28 and a pair of
flashed opal diffusion filters 30 and a photomultiplier tube 32.
Referring to FIG. 6, the basic detector assembly 22 is modified by the
introduction of a dielectric interference filter 34 designed to transmit forward
scatter. The critical angle of the interference filter 34 is matched to the
emission cone of the optical fibers, thus eliminating primary light and
accepting forward scatter. The interference filter 34 can also be replaced by
80
PAGE 9
Xxx. No. 5037207, *
a Xxxxx Diffraction filter which allows for 100% transmission of light with the
exception of a narrow band. The Xxxxx Diffraction Filter has a 10< - 16 >
blocking ability.
When it is desirable to image both fluorescence and forward light scatter the
standard blunt-cut fiber faceplate is replaced by a bias-end cut optical fiber
faceplate 36 and the interference or dielectric filter 34 as shown in FIG. 6.
When it might be desirable to capture images based on optical density and
forward light scatter, the interference filter 34 can be placed between the
standard blunt-cut fiber faceplate 24 of FIG. 5 and the internal reflecting tube
28. The addition of the interference filter 34 permits the imaging of forward
light scatter with both types of optical fiber faceplates.
Referring to FIG. 7, the concept of using the interference filter 34 to
detect forward light scatter is shown. The interference filter 34 can be
optically tuned to detect forward scatter of a variety of discrete images having
discrete ranges of forward scatter angles. In the special instance where the
"exclusion" cone determined by the critical angle of the interference filter 34
exceeds the maximum emission angle of the optical fibers, no light of the
primary wavelength passes through to the photomultiplier tube 32. If the
interference filter 34 is designed to block only the wavelength of the laser
beam and pass other wavelengths, the imaging of laser-excited fluorescence from
the scanned target is possible. With the bias-end cut fibers the only light that
is propagated to the PMT is scattered laser light from the target and
fluorescence emission. Selection of scatter versus fluorescence signals is then
determined by the variable diffraction filter characteristics. The forward light
scatter angle window is variable and determined by the combination of fiber NA
and the characteristics of the filter incorporated in the assembly.
For the special instances of fluorescence shadowing and forward scatter
fluorescence shadowing, the detector assembly 22 is further modified by placing
a fluorescence sensor between the interference filter 34 and the diffusion
assembly 26. For the process of fluorescence shadowing the interference filter
34 is tuned to pass densitometric images to the fluorescence sensor consisting
of a material such as dyed glass which fluorescence at wavelengths accepted by
the photomultiplier tube 32. Images are formed by placing the densitometric
target over the detector assembly 22 and scanning the target. When it is
desirable to perform the process of forward scatter fluorescence shadowing, the
interference filter 34 is tuned to pass forward scatter images onto the
fluorescent sensor which emits fluorescent signals to the PMT 32. The process of
forward scatter fluorescence shadowing has achieved detection resolution of
81
submicron particles in the target in the 0.2-1.0 um range.
It may be desirable to insert an F-theta collimating lens (not shown) between
the beam controller 18 and the target plane 20. Such use of an F-theta lens will
eliminate the changing angle of incidence of the beam of light 16 and direct the
beam in a perpendicular orientation to the target plane 20 to ensure the
accurate propagation of forward light scatter. The Z axis of the beam controller
18 can be preprogrammed to automatically correct for the predictable lack of
flatness in the lens field and the resulting non-perfect image.
An alternative design of the optical fiber faceplate for use in the detection
of fluorescence incorporates square optical fibers instead of round optical
fibers. Square optical fibers can be used to propagate plane-polarized light by
the phenomenon of fluorescence anisotropy.
82
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Xxx. No. 5037207, *
The phenomenon of fluorescence anisotropy is the degree to which the
fluorescence emission from a target is polarized relative to polarization of the
excitation light. Measurement can be accomplished by simply determining the
relative intensities of fluorescence emission with electric field vectors
parallel and perpendicular to the electric field vector of the incident
excitation light. A typical design of an instrument for such a purpose is
presented in a paper by X. Xxxxxxxxx, X. Xxxxxx, Y. Ido, and X. Xxxxxxx,
published in Cytometry, Vol. 8, pp. 35-41, 1987. These authors define
anisotropy, r, as
r = (I = - I + )/(I = + 2I + )
where I = and I + are the fluorescence intensities with electric field
vectors parallel and perpendicular to the electric vector of the incident
excitation light.
The improved method makes use of the fact that square optical fibers having
light absorbing cladding (i.e., extramural light absorbing material or E.M.A.)
possess an unusual property when used to propagate plane-polarized light. As the
plane of polarization of input light rotates about the axis parallel to the
direction of propagation, the plane of polarization of light exiting the fibers
also rotates as expected. However, unlike circular cross section optical fibers,
an additional rotation is observed between plus and minus 20-25 degrees. This
same phenomenon is observed using fibers without E.M.A., however, the range of
differential rotation imparted on the emerging light is reduced to approximately
+ / - 9 degrees.
In effect, the use of square optical fibers with E.M.A. provides a novel
means of amplifying a rotation in fluorescence emission polarization, or the
degradation of polarization relative to excitation light. This is a consequence
of the fact that the square optical fibers tend to rotate the plane of
polarization in the direction of a plane parallel with either diagonal inscribed
within the square core geometry in a manner illustrated in FIG. 8. Therefore, if
incident fluorescent light is rotated (e.g., + 7 degrees relative to the
excitation beam), the emergent light from the fiber optics will be rotated
approximately + 17 degrees as illustrated in FIG. 9. This additional rotation is
imparted as a result of the square cross section geometry and is enhanced by the
presence of E.M.A.
Referring now to FIGS. 10A and 10B, a schematic block diagram of the computer
support system is shown. The processor board is based on the Motorola 68020 32
83
bit microprocessor and the 68881 floating point co-processor. It provides data
collection at a burst rate of one and a quarter million 12 bit words per second
with 256K bytes on board memory accessible with no wait states. A dual-ported
memory card, designed to support the image updating via a random port while the
display RAM features one megabyte dual-ported memory utilizing 32 64K x 4 video
RAM chips, two bit error detection and one bit error correction circuitry is
provided. The sequential (video) port features a row-access to the video memory
providing four 1024 pixels/line data in the internal buffer. With a newly
designed VMEbus display controller board, several display options are provided
for in the system: any continuous 8 bit display out of 16 bit video information
available per pixel is under software control; two display formats are provided,
512 x 480 and 1024 x 960; B/W and color (true and pseudo) displays are provided;
pixel resolution is user selectable 8 to 24 bits; zoom and scroll.
84
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Xxx. No. 5037207, *
Referring to FIGS. 12-17, the image memory controller board is VME bus
compatible. VME bus is a trademark of Motorola, Inc., and is also known as the
IEC821 bus or the IEEE Pio14/DI.O. The VME bus defines an interfacing system
used to connect data processing, data storage, and peripheral control devices in
a closely coupled hardware configuration. The image memory controller board
enables 8, 16 and 32 bit data bus structures. The controller board offers the
following functions. A power-up SCRUB function is provided to fill the memory
array with Hex value FF. This function is not performed when the board is used
as a display memory. The SCRUB is used when the board is used as a conventional
memory. An address range-select function is provided and is based on 1M byte
blocks located on 1M boundaries. For the 32 bit address configuration the upper
8 bits are fixed in a user programmable PAL. This allows for 16 switch
selectable 1M byte ranges using the next lowest four bits. The 1M byte memory is
located on a daughter card and is divided into four banks of 256K bytes each.
Four fully decoded input/output ports are provided. The input/output ports are
located in short address range only (the lower 16 bits), the upper 8 or 16 bits
are fixed. The input/output ports provide the following various support
functions: they enable and disable the EDC circuitry; they report erroneous
addresses; and, they enable and disable the interrupt, overflow, single bit
error, multibit error, register clear, bus error, system failure, and diagnosis
functions. The memory can interrupt any processer card on the bus if there is a
single bit error, multibit error or overflow, provided that the corresponding
enabling bit has been activated. This is a vector interrupt (i.e., a vector will
be supplied to the host processer during the interrupt acknowledge cycle). The
interleaved memory access is supported. Such support makes it possible for 2
memory cards to supply 16 bits two bytes per pixel. All even bytes of the pixel
are stored in one memory card and all odd bytes are stored in the other memory
card.
Referring to FIGS. 18-33, the image memory array board connects to the image
memory controller board by way of two 60 pin interboard connecters. The image
memory array board contains 1M byte of memory divided into four banks. Each bank
consists of 8 dual ported video DRAM (Dynamic Random Access Memory) chips (uPD
41264-15). The random access port of the memory is intended for use by the VME
bus (processer or DMA). The sequential port is dedicated to the video display
circuit. This is an 8 bit per pixel display system although 32 bits are read at
a time from the video memory through the sequential port. A shift register
stores the 32 bits that are read and then supplies them in groups of eight.
Referring to FIGS. 24-26, the CRT controller board is also VME bus compatible
and has a 16 bit data bus structure accessible through a set of input/output
address locations. A 512K byte graphic memory is provided. This memory is
85
organized as a 1K x 1K, 4 bit pixel memory. This organization allows for a color
graphics plane where each pixel may have one of eight colors. This design
supports two dimensional scrolling, zooming and graphic drawing. The CRT
controller board functions as follows. At power up the controller is configured
for no scrolling or zooming and the graphics plane is disabled. The input/output
address is selectable from several ranges. The bit-0 of each graphics nibble is
used to enable or disable an individual pixel. The remaining 3 bits are used to
turn on or off the 3 primary colors of the color monitor system. This provides
for 8 color combinations per pixel. Each color channel is driven by a
digital/analog converter with an internal look-up table (256 x 8 bits). This
table can be used for pseudo color displays of the image stored in memory or for
certain primitive image processing algorithms. The designed display system board
provides 8 bits per channel. Up to three 8 bit image plane boards may provide
the input to the display system board. Therefore, 1-3 image plane boards may
86
PAGE 12
Xxx. No. 5037207, *
be used with a single display system board. The three cases are treated as
follows: in an 8 bit black and white system using one image plane board the same
8 bits are used as input to the look-up tables of the red, green and blue D/A
converters. If two image plane boards are used, the 16 bits per pixel and
special programming hardware is provided to allow the user to chose any
contiguous 8 bits for display. In the case of the three image plane boards, the
overall system becomes a true color system with each image plane board providing
its 8 bits to an individual color channel.
Vertical scrolling may be achieved on an every other line basis. The system
is designed for interlace display and horizontal scrolling is provided for every
4 pixels due to the imaging memory organization. Image zooming is accomplished
by duplication of pixels. The zooming factor is from 2 to 16. Graphics drawing
is supported directly by a Hitachi video display controller (HD63484-8). Various
drawings such as line, circle, ellipsis, etc., are command driven.
Referring to FIGS. 27-29, the graphics memory array board is connected to the
CRT controller board by way of two 60 pin interboard connectors. The graphics
memory array board consists of 512K bytes of DRAM memory for the graphics
display. This board accepts three 8 bit video data channels, displaying the
information on a CRT by using three AM8151 video D/A converters. A circuit is
provided to handle any of the three special cases regarding the number of video
channels. A combination circuit is provided which mixes the video (image) and
graphics display information together for display. Both the video (image) and
graphics can be enabled/disabled without interfering with each other.
The above description of the preferred embodiment is intended for
illustrative purposes and is not intended to be limiting upon the scope and
content of the following claims.
CLAIMS: We claim:
[*1] 1. An improved laser imaging apparatus comprising, in combination:
means for generating a coherent beam of light;
means for securing a target in a fixed position;
a beam controller for receiving and directing such coherent beam of light
into the target in a desired position to position pattern and at a desired rate
of travel;
87
control means for determining the pattern and rate of travel of such beam;
an optical fiber faceplate having a selected emission cone for collecting
light emission transmitted from such target;
a dielectric interference filter having a critical angle matched relative to
such emission cone of said optical fiber faceplate for receiving and blocking
such beam of light and accepting all forward scatter from said faceplate;
means for receiving and filtering such forward scatter from said dielectric
filter;
88
PAGE 13
Xxx. No. 5037207, *1
means for receiving such filtered forward scatter and generating signals as a
function of the intensity and location of the forward scatter received; and
means for receiving such signals and generating a visual display as a
function of the intensity and location of such light emission.
[*2] 2. The imaging apparatus of claim 1, wherein said dielectric
interference filter is a Xxxxx Diffraction filter.
[*3] 3. The imaging apparatus of claim 1, wherein said beam controller has X,
Y coordinate scanning capability with a simultaneous Z coordinate correction to
cause such beam to have a topographical field of focus in the plane of the
target.
[*4] 4. An improved laser imaging apparatus comprising, in combination:
means for generating a coherent beam of light;
means for securing a target in a fixed position;
a beam controller for receiving and directing such coherent beam of light
into the target in a desired position to position pattern and at a desired rate
of travel;
control means for determining such pattern and rate of travel of such beam;
a blunt-cut optical fiber faceplate having a selected emission cone and
having a selected acceptance angle of a high numerical aperture to capture
differences in the optical density of such light emission being transmitted from
the target;
a dielectric interference filter having a critical angle matched relative to
such emission cone of said optical fiber faceplate for accepting and
transmitting light emission based on optical density and forward scatter;
means for receiving and filtering such light transmitted from said dielectric
interference filter;
means for receiving such filtered light and generating signals as a function
of the intensity and location of the light received; and,
89
means for receiving such signals and generating a visual display as a
function of the intensity and location of such light emission.
[*5] 5. The imaging apparatus of claim 4 further including a fluorescent
sensor located between said dielectric interference filter and said filter
means, said fluorescent sensor receiving light emission from said dielectric
interference filter and creating fluorescent shadow images from said light
emission.
[*6] 6. The imaging apparatus of claim 4, wherein said beam controller has X,
Y coordinate scanning capability with a simultaneous Z coordinate correction to
cause such beam to have a topographical field of focus in the plane of the
target.
90
PAGE 14
Xxx. No. 5037207, *6
[*7] 7. An improved laser imaging apparatus comprising, in combination:
means for generating a coherent beam of light;
means for securing a target in a fixed position;
a beam controller for receiving and directing such coherent beam of light
into the target in a desired position to position pattern and at a desired rate
of travel;
control means for determining such pattern and rate of travel of such beam;
an optical fiber faceplate having a selected emission cone for collecting
light emission transmitted from the target, said optical fibers of said
faceplate being of a square cross section to propagate plane polarized light
through fluorescence anisotropy;
filter means for receiving and filtering such light collected by said
faceplate;
means for receiving such filtered light and generating signals as a function
of intensity and location of the light received; and,
means for receiving such signals and generating a visual display as a result
of the intensity and location of such light emission.
[*8] 8. The imaging apparatus of claim 7, wherein said cross sectional
optical fibers include extramural light absorbing material, said material
enhancing the ability of said square optical fibers to propagate plane polarized
light.
[*9] 9. The imaging apparatus of claim 7, wherein said beam controller has X,
Y coordinate scanning capability with a simultaneous Z coordinate correction to
cause such beam to have a topographical field of focus in the plane of the
target.
[*10] 10. An improved laser imaging apparatus comprising, in combination:
means for generating a coherent beam of light;
means for securing a target in a fixed position;
91
a three-dimensional beam controller for receiving and directing such coherent
beam of light into the target in a desired position to position pattern and at a
desired rate of travel, said beam controller having X, Y coordinate scanning
capability with a simultaneous Z coordinate correction to cause such beam to
have a topographical field of focus in the plane of the target;
control means for determining the three-dimensional pattern and rate of
travel of such beam;
a bias-cut optical fiber faceplace having a selected acceptance angle to
block any light being transmitted from the target which is outside of such
acceptance angle and capture any other light emission from the target which is
within such specified acceptance angle;
92
PAGE 15
Xxx. No. 5037207, *10
a dielectric interference filter having a critical angle matched relative to
said selected acceptance angle of said bias-cut optical fiber faceplate to
accept and transmit submicron light emission based on fluorescence and forward
light scatter while blocking such beam of light;
means for receiving and filtering such submicron emission from said
dielectric interference filter;
means for receiving such filtered submicron emission and generating signals
as a function of the intensity and location of the fluorescence and forward
light scatter comprising such submicron emission; and
means for receiving such signals and generating a visual display as a
function of the intensity and location of such images.
[*11] 11. The imaging apparatus of claim 10 further including a fluorescent
sensor for receiving such fluorescence and forward scatter submicron emission
from said dielectric interference filter and creating fluorescent shadow images
in the 0.2-1.0 um range.
93
FIGURES TO PATENT NO. 5,037,207
94
[FIG. 1 is a graph showing the relationship of the numerical aperture (NA) and
target field image size for conventional lenses.]
95
[FIG. 2 is a schematic illustration of the laser imaging system of the present
invention.]
96
[FIG. 3 is an illustrative representation of a typical microscope slide and a
pattern of laser scanning the slide.]
97
[FIG. 4 is an illustrative representation of the light detection properties of a
blunt-cut optical fiber faceplate and bias-cut optical fiber faceplate.]
98
[FIG 5. is a schematic illustration of a typical detector assembly as used with
the present invention.]
99
[FIG. 6 is a schematic illustration of a detector assembly for use with the
present invention in detecting fluorescence and forward light scatter.]
100
[FIG. 7 is a conceptical illustration of the detector assembly of FIG. 6.]
101
[FIG. 8 is a graph showing optical fiber polarimetry for square cross-sectional
optical fibers.]
102
[FIG. 9 is an illustrative schematic of the plane polarization by a square
cross-sectional optical fiber of fluorescence emissions.]
103
[FIG. 10A is a schematic block diagram of the image memory controller and image
memory array for the imaging system of the present invention.]
104
[FIG. 10B is a schematic block diagram of the CRT controller and graphics memory
array for the imaging system of the present invention.]
105
[FIG. 11 is a schematic illustration of the light acceptance cone of a bias-cut
optical fiber faceplate.]
106
[FIGS. 12A, B, C and D are schematics for the Image Memory Controller of the
present invention showing the circuitry for the Memory Request Arbiters and the
Input/Out Access circuitry.]
107
[FIGS. 13A, B, C and D are schematics for the Image Memory Controller of the
present invention showing the circuitry for the Memory and the Input/Output
Address Decoder.]
108
[FIGS. 14A, B, C and D are schematics for the Image Memory Controller of the
present invention showing the circuitry for the Memory Refresh Function and the
Interrupt Request Function and Data Transfer Acknowledgment Generation.]
109
[FIGS. 15A, B, C and D are schematics showing the Address Multiplexer circuitry
for the Image Memory Controller of the present invention.]
110
[FIGS. 16A and B are schematics showing the Memory Buffers circuitry for the
Image Memory Controller of the present invention.]
111
[FIGS. 17A and B are schematics showing the Input/Output registers circuitry for
the Image Memory Controller of the present invention.]
112
[FIGS. 18A, B, C and D through 20A, B, C and D are schematics showing the Memory
Chip Array circuitry for the Image Memory Array of the present invention.]
113
[FIGS. 21A, B, C and D are schematics showing the Memory Arrays, Address liners,
and Control Bus circuitry for the Image Memory Array of the present invention.]
114
[FIGS. 22A, B, C and D are schematics showing the Image Data Shift Register
circuitry for the Image Memory Array of the present invention.]
115
[FIG. 23 is a schematic showing the Memory Array circuitry for the Image Memory
Array of the present invention.]
116
[FIGS. 24A, B, C and D are schematics showing the Video Display Generator
Interface circuitry for the CRT controller of the present invention.]
117
[FIGS. 25A, B, C and D are schematics showing the Address Decoder, Graphics
Memory Interface and Interrupt Request and Acknowledge circuitry of the CRT
Controller of the present invention.]
118
[FIGS. 26A, B, C and D are schematics showing the Zoom Control and Look-up Table
Circuitry for the CRT Controller of the present invention.]
119
[FIGS. 27A, B, C and D are schematics showing the Memory Chip Array circuitry
for the Graphics Memory Array of the present invention.]
120
[FIGS. 28A, B, C and D are schematics showing the Graphics Display and Shift
Register circuitry for the Graphics Memory Array of the present invention.]
121
[FIGS. 29A, B, C and D are schematics showing the D/A Converter circuitry for
the Graphics Memory Array of the present invention.]
122
Exhibit C2 to Xxxxxx-Xxxxx License Agreement
123
US PATENT 4,877,966
<=2> GET 1st DRAWING SHEET OF 14
Oct. 31, 1989
Method and apparatus for the measurement of low-level
laser-induced fluorescence
INVENTOR: Tomei, L. Xxxxx, Dublin, Ohio
Cornhill, Xxxx, Worthington, Ohio
Xxxxxxxxx, Jogikal, Columbus, Ohio
Xxxxxxxx, Xxxxxxx, Columbus, Ohio
DISCLAIMER: Jul. 19, 2005
ASSIGNEE-AT-ISSUE: Ohio State University Research Foundation, Columbus, Ohio
(02)
APPL-N0: 150,293
FILED: Jan. 29, 1988
REL-US-DATA:
Division of Ser. No. 828,694, Feb. 12, 1986
INT-CL: [4] X00X 0#00
XX-XX: 250#458.1; 250#461.2
CL: 250
SEARCH-FLD: 250#458.1, 459.1, 461.2, 461.1; 350#96.27, 527
REF-CITED:
U.S. PATENT DOCUMENTS
3,450,455 6/1969 * Xxxxxx
3,516,746 6/1970 * Shibata et al.
3,981,590 9/1976 * Xxxxxxx
4,107,534 8/1978 * Piltingsrud 25#368
4,523,799 6/1985 * Delhaye 250#527
4,758,727 7/1988 * Tomei et al. 250#458.1
124
FOREIGN PATENT DOCUMENTS
0123942 7/1984 * European Patent Organization 250#327.2
0155813 9/1985 * European Patent Organization 250#368
OTHER PUBLICATIONS
Bussini, et al., "A Silicon Rubber Scintillation Compound for Complex Geometry
Radiation Detectors", Nuclear Instruments and Methods, No. 2, (1973) pp.
333-335.
Xxxx, et al., "Vidicon Microscope for Counting Fluorescent Particles", Review
125
PAGE 17
Xxx. No. 4877966,*
of Scientific Instruments, vol. 42, No. 4 (1971) p. 508.
Xxxxxx, F., "Mural Television Display Using Fiber Optics", RCA Technical Notes
(RCA TN No. 188 (1958).
"The FACS Analyzer Fluorescence and Volume Cell Analysis" Brochure, Becton
Xxxxxxxxx FACS System, 1982.
"The New FACS Series, FACS 440, FACS 420, FACS 400, Fluorescence Activated Cell
Sorting", Becton Xxxxxxxxx FACS Systems, 1981.
"Photodigitizing with PDS Microdensitometer Systems" Brochure, Xxxxxx-Xxxxx,
not dated.
"Microdensitometer 3CS" Brochure, Xxxxx-Xxxxx, not dated.
"Microdensitometer 6" Brochure, Xxxxx-Xxxxx, not dated.
"EIKONIX Registered TM Applied Research Software and Product Development
Feasibility and Impact Studies Automated Instruments" Brochure, EIKONIX
Registered TM Corporation, not dated.
"ACAS 470 Workstation" Brochure, Meridian Instruments, Inc., not dated.
PRIM-EXMR: Xxxxxx, Xxxxxx X.
ASST-EXMR: Xxxxx, Xxxxxxx
LEGAL-REP: Emch, Schaffer, Xxxxxx & Xxxxxxxx Co.
ABST:
A method and apparatus for measuring low-level laser-induced fluorescence are
disclosed. A laser is used to produce a coherent beam of light which is
sequentially passed through a beam expander, iris diaphragm, focusing lens and
three-dimensional scanner before being focused onto a rigidly mounted target. A
computer is used to predeterminately control the pattern and the rate at which
the scanner passes the beam of light over the target. The light transmitted onto
the target induces fluorescent light in the target. The fluorescent light is
sequentially gathered by a biased cut optical fiber member and directed into a
photomultiplier tube where the intensity of the fluorescent light is measured.
The intensity data is then digitized and recorded by the computer as a function
of the coordinates of each preprogrammed point location of the beam impinging
upon the target. This data is used to produce an image of all or a portion of
the target on a visual monitor. In addition, the data can be recalled and used
for further analysis of the target.
NO-OF-CLAIMS: 26
EXMPL-CLAIM: <=9> 1
126
NO-OF-FIGURES: 18
NO-DRWNG-PP: 14
PARCASE:
This is a divisional of copending U.S. application Ser. No. 828,694 filed on
2/12/86.
SUM:
127
PAGE 18
Xxx. No. 4877966, *
BACKGROUND OF THE INVENTION
The present invention relates a method and apparatus for the measurement of
low-level laser-induced fluorescence in the field of cytofluorometry.
Cytofluorometry has been greatly enhanced with the development of fluorescence
dyes that are specific for deoxyribonucleic acid (DNA). These dyes permit the
determination of the ratios of total DNA present in a cell population and, since
the total amount of DNA doubles as a cell progresses through its proliferation
cycle, the distribution of cell cycle positions that existed in a cell
population is easily determined statistically. This is based upon the fact that,
within necessary tolerances, the amount of dye bound to a cell is directly
proportional to the amount of DNA present.
Fluorescence is the emission of light whose wavelength is different from that
used to induce or excite the molecule of dye. Therefore, common fluorescence
techniques require that the dye bind specifically to some component which is to
be measured such as DNA. Such specificity is obtained by structurally modifying
the dye molecule or coupling a dye molecule to another molecule that has the
required specificity of binding. Another technique which utilizes fluorescence
emission is based upon other properties of some dye molecules. When these
molecules are in an aqueous environment, their fluorescence characteristics are
distinctly different from that obtained in a hydrophobic environment. Since all
biological membranes have hydrophobic regions, the amount and kind of
fluorescence obtained following staining is related to the structural state of
that membrane.
Several analytical techniques have been developed which exploit these various
properties of fluorescent dyes. These techniques include photobleaching,
fluorescence quenching, and shifts in fluorescence emission spectra. Problems
are continuously encountered with these various techniques in that total
fluorescence per cell, following any general technique with any particular dye,
varies markedly and the variation is not quantitated for the expressed purpose
of defining the degree or nature of cell cooperativity in a coordinating,
interacting, cell mass.
Another fluorescence technique, stereological computer assisted
cytofluorometry (SCAC) provides for the measurement of laser-induced cellular
fluorescence in a cell or tissue mass (e.g. monolayer cell cultures or tissue
sections). Dependent upon the nature of the fluorescent dye employed, the
cellular response-density distribution profile will provide data of profound
theoretical as well as practical significance. SCAC provides a technique for the
128
quantitation of cell behavior and responses to drugs within the context of the
cell mass or tissue. This has led to the development of new pharmacological
parameters and is expected to lead to more refined and sophisticated parameters
with which to study drug actions in the fields of cancer diagnosis,
chemoprevention and chemotherapy, environmental and forensic toxicology, as well
as basic biological sciences.
Currently there are two basic apparatuses for performing cytofluorometric
studies. A fluorescence microscope such as the FACS TM series analyzer
manufactured by Becton Xxxxxxxxx provides observation of cells contained in a
culture dish. The fluorescence microscope is highly accurate when analyzing a
small area but cannot measure cell groupings larger than a culture dish without
destroying the spatial relationship of cells. The second apparatus, a Flow
Cytometer is designed to provide observation for a large number of cells, but
129
PAGE 19
Xxx. No. 4877966, *
the cells must be in suspension, thereby eliminating any possibility of
obtaining spatial data. The CYTOFLUOROGRAF system from the Ortho Instruments
Corporation is an example of a flow cytometer. The flow cytometer provides
highly accurate information on the frequency distribution of fluorescence
intensity in a randomly dispersed cell populations. However, no information is
provided regarding the spatial relationship that may exist between cells in the
tissue, tumor or culture prior to dispersal and staining.
Interpretation of frequency distributions of fluorescence intensity is
seriously hampered by the fact that the response of the original cell population
is rarely spatially homogenous. Heterogeneity of cell identity, morphology and
drug responsiveness is commonly observed but not considered in current
cytofluorometric analytic techniques. However, population heterogeneity is
regarded by biologists as being an inherent quality of coordinating cell
populations found in all animal tissues, tumors, primary cell cultures, and in
rudimentary form, laboratory cell lines. The present invention, method and
apparation, provides the ability to observe large areas of tissue, while
maintaining full spatial relationships without the need for any special
preparation.
SUMMARY OF THE INVENTION
The present invention relates a method and apparatus capable of rapid
wide-field scanning of low-level laser induced fluorescence of tissue sections,
cell cultures, and other biological materials, while maintaining high spatial
resolution. The method and apparatus of the present invention measures off-axis
fluorescence. The fluorescence measurements permits full digitalization of
images with 16 bit precision for 2-4 x 10<6> pixels. The target area scanned
are greater than 25 cm<2> with a resolution of 5-10 mu m and the scanning time
is between 1 and 6 seconds dependent upon computer processing and storage
limitations. The SCAC methodology, provides for the acquisition of information
from a large number of individual cells as does flow cytofluorometry. However,
in addition to individual cell responses, the present invention will provide
highly accurate information regarding the spatial distribution of those cells
within the total population. This is of particular importance in the areas of
tumor biology, pathology and early detection of abnormal cells in tissues and
organs. The SCAC design of the present invention provides for analysis of large
cell numbers in vitro with high spatial precision.
The apparatus of the present invention incorporates an optical fiber taper
for high efficiency light gathering and transmission to a highly sensitive
130
detector. The properties of the fibers can be exploited to shift the detector
offaxis. This apparatus of the present invention makes use of two fiber
properties: tapered fiber transmission and biased cut deflection. Further,
extra-mural absorption material are added to provide further attenuation of
off-axis incident laser light. The method and apparatus of the present invention
will be further understood by the following description of the preferred
embodiment with reference to the following figures.
DRWDESC:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a block diagram of a scanning device according to the present
invention.
131
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Xxx. No. 4877966, *
FIG. 2 is an exemplary view of a scanning pattern of a translucent object
with a scanning device according to the present invention.
FIG. 3 is a block diagram of the electronic interface used to control the
various components of a scanning device according to the present invention.
FIG. 4 is a block diagram showing an alternate embodiment of the fiber optic
bundle as used with a scanning device according to the present invention.
FIG. 5 is a flowchart representing the scanning program by which the present
invention operates.
FIG. 6a is an exemplary view of a biased cut optical fiber faceplate.
FIG. 6b is an exemplary view of a biased cut tapered optical fiber faceplate.
FIG. 7 is a schematic showing an analogue to digital convertor board as used
in the present invention.
FIG. 8 is a schematic showing the clock circuitry as used in the present
invention.
FIG. 9 is a schematic showing the low-order Address but Interface circuitry
as used in the present invention.
FIG. 10A, B is a schematic showing the DMA/Versabus Interface circuitry as
used in the present invention.
FIG. 11 is a schematic showing the DMA data path circuitry as used in the
present invention.
FIG. 12 is a schematic showing the timer interface circuitry as used in the
present invention.
FIG. 13a is a schematic showing the 1.5 volt local voltage source as used in
the present invention.
FIG. 13b is a schematic showing the Power-On Clear circuitry as used in the
present invention.
FIG. 14A, B is a schematic showing the board select logic as used in the
132
present invention.
DETDESC:
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention relates to a method and apparatus for the off-axis
detection of laser-induced fluorescence. Referring to FIG. 1, there is shown a
block diagram of the components of a scanning device 10 according to the present
invention. The scanning device 10 uses coherent light to scan a object or target
12. In the preferred embodiment, the components of the device 10 include; a
tuneable laser 14, a beam expander 16, an iris diaphragm 18, a focusing lens 20,
a three-dimensional scanner 22, a target mount 24, a fiber optic faceplate
having a bias cut 26, a diffusion member 28, a photomultiplier tube 30, a
133
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Xxx. No. 4877966, *
programmable computer with memory 32 and a visual monitor 34. Together, these
components provide for rapid scanning of translucent targets to yield high
resolution, wide-field fluorescence analyses of a large number of cells in
vitro, with high spatial precision.
The means for generating the coherent beam of light is a laser 14. It is
desirable that the laser 14, shown in FIG. 1 emit a coherent polarized beam of
light 15 having a wavelength in the range of 350 to 540 nanometers. The light
beam 15 exiting the laser 14 is approximately 1 millimeter in width, which is
too small to be used effectively by the reflecting mirrors within the scanner
22. Therefore, the present invention provides a beam expander 16 which receives
the laser beam 15 and enlarges the size of the beam 15 to ensure utilization of
the greatest possible surface area of the reflecting mirrors within the scanner
22. The preferred beam expander 16, as shown in FIG. 1, is a Buschnell variable
beam expander having a magnification power range of 9 x to 30 x . In the
preferred embodiment, the beam expander 16 is adjustable to expand the beam size
through a range from 1 millimeter to 10 millimeters.
As an alternative to the use of a variable beam expander, a single power beam
expander may also be used with Applicants' invention. In such cases an iris
diaphragm 18 is included to receive the expanded beam 15 from the single power
beam expander. The iris diaphragm 18, also known as a field iris, is intended to
vary the diameter of the beam 15 input to the focusing lens 20 and the scanner
22, thus varying the spot size on the target 12. Consequently, the iris
diaphragm 18 is used to more finely tune the size of the beam of light 15.
Referring now to FIG. 1, the beam of light 15 is exiting the beam expander 16
or iris diaphragm 18 is passed through the focusing lens 20 which focuses the
beam of light 15 on the target 12. Together, the beam expander 16/iris diaphragm
18 and focusing lens 20 permit the beam of light to be focused to a spot of
predetermined dimension at the target plane of the translucent target 12.
In the preferred embodiment, the light received by the focusing lens 20 is a
gaussian columnated coherent beam of light. Using this assumption, the final
spot size upon the target 12 is diffraction limited and governed by the
following equation:
d = 4F lambda / pi D, TM (1)
,
134
wherein
d = spot size
F = focal length of focusing lens
lambda = wavelength of the light
D = aperture of input beam to focusing lens
According to equation (1), the proper selection of the focal length (F) of
the focusing lens 20, the wavelength ( lambda ) of the coherent light beam 15,
and the aperture (D) of the input beam to the focusing lens 20, will allow for
the size of the beam spot (d) on the target 12 to be varied as desired. The
135
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Xxx. No. 4877966, *
smallest available spot size is approximately one micron. Thus, the present
invention is capable of scanning at a variety of intervals with the smallest
being a one micron interval.
The focused beam of light 15 exiting the focusing lens 20 is received by the
three-dimensional scanner 22 which operates to control the passing of the light
beam 15 over the target 12. The scanner 22 as shown in FIG. 1 is manufactured by
General Scanning, Inc. and uses computer controlled mirrors to pass the focused
beam of light 15 back and forth across the target 12. The scanner 22 is capable
of scanning an area of any size, up to 20 millimeters by 40 millimeters, while
operating at a raster rate of up to 20 Hz or a vector rate of up to 280 mm/sec.
Computer controlled drive motors cause the mirrors of the scanner 22 to move
such that the scanner 22 has accurate X and Y coordinate scanning capabilities
with a simultaneous Z coordinate correction to yield a beam of light 15 with a
flat field of focus. If desired, the Z coordinate of the scanner 22 can be
programmed to coordinate with an uneven topographical field of focus on the
target 12.
The scanner 22 passes the focused coherent beam of light to the target 12,
thereby inducing fluorescence in a predetermined pattern and at a predetermined
rate set by the computer 32 so that data is obtained as to the intensity of the
fluorescence at differing points on the target 12. At precise predetermined
points during the scanning process, the intensity of the light is measured by
the photomultiplier tube 30 and recorded by the computer 32 as a function of
location of the beam 15 on the target 12. By taking successive point
measurements of the intensity of the fluorescence and relating them back to the
location data in the computer 32, the data is used to analyze the target 12 or
recreate a visual image of the target 12 on a visual monitor 34.
The target 12 is scanned over a very large series of points using a back and
forth pattern as shown in FIG. 2. The X, Y coordinates and the rate of scanning
are predetermined and programmed through the computer 32. The scanning program
is individually drawn to the specifics of the application. The flowchart for the
scanning program is shown in FIG. 5. At each scanning point, the fluorescence
intensity is measured and recorded as a function of the X, Y coordinates of the
beam. For example, the scanner 22 would be programmed to scan the target 12, as
shown in FIG. 2, in a sequential pattern over the following (X,Y) coordinates:
(1,1), (1,2), (1,3), (1,4), (2,4), (2,3), (2,2), (2,1), (3,1), (3,2), (3,3),
(3,4), (4,4), (4,3), (4,2) and (4,1). The intensity of the fluorescence being
excited at each of the scanning points is measured by the photomultiplier tube
30 in terms of analogue data. The analogue data for each scanning point is then
136
digitized by the computer 32 and recorded as a function of the X,Y coordinates
of that particular scanning point.
The present invention utilizes a three-dimensional scanner, such as that
manufactured by General Scanning, Inc. The use of such a scanner provides in
that the target mount 24 is stationary, thereby eliminating wobble and jitter
experienced with moving target mounts. Due to the minute size of the focused
beam spot on the target 12 and the large amount of point data being taken, it is
important that the target 12 be rigidly mounted in a fixed position on the
target mount 24 to maintain the proper focusing, depth of field, and spot size.
Positioned directly below the target mount 24 and thus below the target 12
itself is a fiber optic faceplate 26 which is used in conjunction with a
diffusion member 28 to increase the efficiency of the highly sensitive
137
PAGE 23
Xxx. No. 4877966, *
photomultiplier tube 30. The sensing surfaces of photomultiplier tubes are
characteristically nonuniform. The fluorescent light transmitted by the target
12 is minute in the form of a narrow columnated beam. If this beam were received
by the photomultiplier tube 30, variations in the recorded intensity of the beam
would occur as a result of variations in the location at which the narrow
columnated light beam strikes the sensing surface of the photomultiplier tube
30. Therefore, the present invention utilizes the fiber optic faceplate 26 to
gather as much light as possible and the diffusion member 28 spreads the light
beam broadly as possible to achieve full use of the photomultiplier tube 30.
One of two types of fiber optic faceplates 26 may be used with the present
invention, depending upon the size of the window of the photomultiplier tube 30.
When the window of the photomultiplier tube 30 is comparable in size to the
target 12, then a flat disk-type faceplate, as shown in FIGS. 1 and 2, is used.
When the scanning area of the target 12 is larger than the window of the
photomultiplier tube 30, a tapered or frustoconical shaped fiber optic faceplate
26A, such as shown in FIG. 4, is used. The use of such fiber optic faceplates
presents a marked improvement over prior art systems in which lenses are relied
upon to gather and direct the light beam. The light gathering or flux-carrying
capacity of the optical fibers within the taper 26A as used with the present
invention is 10 to 70 times higher than that of standard optical lenses. The
relative increase in light gathering capacity is numerically equal to the ratio
of the squares of the numerical apertures. The effective numerical aperture of a
typical lens capable of imaging a target area of at least several square
millimeters is 0.10 to 0.20 as compared with optical fibers which have nominal
numerical aperture values of greater than 0.60. Therefore, the use of optical
fibers in conjunction with highly sensitive photomultiplier tubes 30 produces a
marked advantage over instrument designs incorporating optical lens systems
and/or solid-state light detectors.
The fiber optic faceplate 26 is biased cut at a angle alpha , preferably 30o.
Referring to FIG. 6, the bias cut on the optical fiber faceplate 26 is shown as
angle alpha and the acceptance angle of the fiber optic faceplate 26 is
deflected by angle beta . This provides a total deflection by angle beta or
shift of the acceptance angle theta which comprises the acceptance cone of the
fiber optic bundle. If laser light is shown onto a fiber outside of the
acceptance cone, the amount of light transmitted along the length of the fiber
will be severely limited. Thus, the present invention provides a fiber optic
faceplate 26 having a bias cut such that light hitting perpendicular to the face
of the fibers will not be in the acceptance cone. In this manner, a light source
hitting perpendicular to the surface will be attenuated while excited
138
fluorescence or scattered light may enter the acceptance cone. The bias cut
fiber optic system of the present invention is capable of gathering 30% of the
excited fluorescence from the target being scanned, as compared with current
technology in which only 2% of the excited fluorescent light is gathered.
The optical fibers in the faceplate 26 also function as the first stage for
light diffusion. Optical fibers will gather light from all angles within the
acceptance cone. Upon exit from the faceplate 26 this light is rotated about a
solid angle through which the intensity is uniformly distributed. This optic
fiber property thus enhances the light scattering efficiency of the diffusion
member 28.
Further attenuation of the light is accomplished by insertion of neutral
density filters 27 between the fiber optic faceplate 26 and the diffusion
139
PAGE 24
Xxx. No. 4877966, *
member 28. In the preferred embodiment, a 620 nanometer filter is incorporated
in order to eliminate the normal fluorescent room lighting, thus permitting
operation of the instrument without extensive light protection. Further, a
barrier filter (not shown) may be incorporated at this point to absorb light at
the excitation wavelength while transmitting light at the fluorescent
wavelength.
The diffusion member 28, shown in FIG. 1, is an open-ended hollow elongated
member with a reflective interior surface. Such a member can be constructed of
an open-end box using mirrored glass for the interior surface. Alternatively,
milk glass may be used as the diffusion member 28. The primary functions of the
diffusion member 28 are two-fold. First, the diffusion member 28 blocks out any
remaining ambient light which will affect the intensity readings of the
photomultiplier tube 30. Secondly, the diffusion member 28 allows the
transmitted light to diffuse within its confines to more fully utilize the
window of the photomultiplier tube 30 and thus yield more accurate results.
After the fluorescent light transmitted from the target 12 is directed to the
photomultiplier tube 30 by the fiber optic faceplate 26 and diffusion member 28,
the photomultiplier tube 30 detects and measures the fluorescence levels. The
photomultiplier tube 30 is in turn linked to the computer 32 so that data
obtained by the photomultiplier tube 30 regarding the intensity of the light is
fed into the computer 32.
Once the device 10 is activated and scanning of the target 12 begins, the
output of the photomultiplier tube 30 is sampled by the computer 32 as often as
desired. The electrical signals relating the intensity of the fluorescent light
are converted from analogue to digital values by the computer 32 for storage
purposes. Using the preprogrammed position data and the stored intensity values,
the computer 32 can provide a realtime pictorial reconstruction of the target 12
on a visual monitor 34. The capacity of the computer 32 to rapidly store the
highly detailed data being viewed makes it possible to depict the entire area of
the target 12 on the visual monitor as well as focus on particular points of
interest by providing enlarged viewing of select portions of the target 12.
Referring now to FIG. 3, an interface is provided to collect the analogue
intensity data output of the photomultiplier tube 30 and convert the data into
digital form for the versabus-based computer 32. The interface 33 communicates
with the computer 32 through direct memory access (DMA) with a minimum
instantaneous transfer rate of 100,000 picture elements (pixels) per second. The
interface 33 of the present invention is composed of five main subsystems
140
including: an analogue-to-digital (A/D) converter 36 as shown in FIG. 7; a set
of buffers and latches 38; bus interface logic 40 as shown in FIGS. 9, 10; a DMA
controller 42 as shown in FIG. 11; and a system timing controller 44 as shown in
FIG. 12. The DMA controller 42 and system timing controller 44 are used to
properly operate the A/D converter 36, the set of buffers and latches 38 and the
bus interface logic 40.
Referring now to FIG. 7, the A/D converter 36 transforms the image data
signals which are received from the photomultiplier tube 30 into a digital form
which is understandable by the host computer 32. The buffers change the voltage
of the signals to a level which is compatible with the host computer 32 while
the latches store the transformed data until it is efficient for the computer 32
to accept them.
141
PAGE 25
Xxx. No. 4877966, *
Referring now to FIG. 11, the DMA controller 42 and the bus interface logic
40 of FIGS. 9 and 10 store and control information about the flow of the data to
the computer 32 so that the computer 32 receives the data only when it is able
to accept the information. The system timing controller 44 of FIG. 12 ensures
that the proper number of pixels are placed into the memory 46 of the computer
32. In addition, the system timing controller 44 generates all the needed
signals for the A/D converter 36.
Since the A/D converter 36 outputs emitter coupled logic (ECL) compatible
signals, level shifters are required to provide transistor-transistor logic
(TTL) signals. These signals are tied directly to a combination bus buffer/latch
chips. The conversion timing is derived from a 1 MHz crystal feeding an AM9513
timing controller. It should be noted that higher frequency crystals may be
used. The timing controller is started by means of an enable convert signal
which can be generated by the host computer 32 under user control, or by a
signal external to the board. The timing controller 44 generates the convert and
latch signal, and also terminates its count automatically. The controller is
completely software programmable.
The bus interface logic permits the use of both a positive true logic and
negative true logic CPU bus. The bus also allows for a choice of an 8 or 16 MHz
CPU clock speed. The board is memory-mapped, and may reside on any 256 byte
boundary in memory.
The DMA controller circuit is designed to provide for both cycle steal and
burst modes, under software programmability. This feature allows for increased
efficiency at high data rates. Regardless of the mode chosen, the eight most
significant bits are latched, and the next sample is latched in the same manner.
At this point, the DMA transfer will proceed in the specified mode. It is
important to note that by first latching 16 bits and then requesting the bus,
the bus bandwidth used in cycle steal mode is cut to one half of what it would
otherwise have been.
The remainder of the circuitry includes a 1.5 volt local power supply 48
(FIG. 13a), as required by the DMA control circuitry, and a dual speed
power-on-clear circuit 50 (FIG. 13b) which included to ensure the circuit powers
up in a predictable and stable manner.
With the circuitry in place, the device 10 is used to scan the object 12. The
excited fluorescent intensity is determined using the highly efficient optical
fiber faceplate 26 and the sensitive photomultiplier tube 30 without respect to
142
the precise position of the laser beam on the target object. Such position is
alternatively determined by the computer 32 which controls the threedimensional
scanner 22. The computer 32 then combines the X, Y coordinate spatial
information with the intensity information to yield a high resolution wide-field
image of the object 12.
Having thus described the invention in detail, it should be understood that
various modifications and changes can be made in the apparatus without departing
from the scope and content of the following claims.
CLAIMS: What we claim:
[*1] 1. An improved method for measuring the excited fluorescence of an
object comprising:
143
PAGE 26
Xxx. No. 4877966, *1
binding a specific dye to such object;
securing such object in a fixed position;
generating a coherent beam of light;
scanning such fixed object with said coherent beam of light in a
preprogrammed three dimensional pattern and at a preselected rate to induce such
fluorescence;
gathering such fluorescent light with an optical fiber member, such optical
fiber member attenuating said coherent beam of light used to induce such
fluorescence;
measuring the intensity of said fluorescent light in a light intensity
measuring device; and
sequentially recording data regarding the intensity of said fluorescent light
as a function of the location of said scanning beam of light on said object.
[*2] 2. The method of claim 1 further including the step of filtering said
fluorescent light upon receipt from said optical fiber member and prior to
measuring the intensity of said light.
[*3] 3. The method of claim 2 further including the step of diffusing said
filtered light upon receipt from said filter and prior to measuring the
intensity of said light.
[*4] 4. The method of claim 1 which further includes the step of producing a
visual image of at least a portion of said object based upon said sequentially
recorded data of the intensity of said fluorescent light as a function of the
location of said scanning beam of light on said object.
[*5] 5. The method of claim 4, wherein said visual image is realtime produced
on a visual monitor.
[*6] 6. The method of claim 1, which further includes the step of focusing
said beam of light through a focusing lens after the step of generating a
coherent beam of light and before the step of directing said beam of light into
said three dimensional scanner.
144
[*7] 7. The method of claim 6, which further includes the step of expanding
said beam of light between the steps of generating and focusing said beam of
light.
[*8] 8. The method of claim 1, which further includes the step of storing
said sequentially recorded data in a retrievable digital form.
[*9] 9. The method of claim 1, wherein a laser is used to generate said
coherent beam of light.
[*10] 10. The method of claim 1, wherein a programmable computer is used to
control the movement of said scanner and record said data regarding the
intensity of said fluorescent light.
145
PAGE 27
Xxx. No. 4877966, *10
[*11] 11. The method of claim 3, wherein an open-ended hollow elongated
member having a reflective interior surface is used to diffuse said fluorescent
light.
[*12] 12. The method of claim 1, wherein said light measuring device is a
photomultiplier tube.
[*13] 13. The method of claim 7, wherein a beam expander is used to expand
said beam of light.
[*14] 14. A method measuring the excited fluorescence of an object
comprising;
binding a specific dye to said object;
securing such object in a fixed position;
generating a coherent beam of light;
expanding said beam of light;
focusing said expanded beam of light;
scanning such fixed object with said focused beam of light in a preprogrammed
three dimensional pattern and at a preselected rate to induce such fluorescence;
collecting such induced fluorescent light from said object by means of a
biased cut optical fiber faceplate such bias cut of such optical fiber faceplate
acting to eliminate any light directed perpendicular to the surface of such
object;
filtering said fluorescent light;
diffusing said filtered light;
measuring the intensity of said diffused light in a light intensity measuring
device; and
sequentially recording data regarding the intensity of said fluorescent light
as a function of the location of said scanning beam of light on said object.
146
[*15] 15. The method of claim 14 which further includes the step of storing
said sequentially recorded data in a retrievable digital form.
[*16] 16. The method of claim 14 which further includes the step of producing
a visual image of at least a portion of said object based upon said sequentially
recorded data of the intensity of said fluorescent light as a function of the
location of said scanning beam of light on said object.
[*17] 17. The method of claim 16, wherein said visual image is produced on
a visual monitor.
[*18] 18. The method of claim 14, wherein a laser is used to generate said
coherent beam of light.
147
PAGE 28
Xxx. No. 4877966, *18
[*19] 19. The method of claim 14, wherein a programmable computer is used to
control the movement of said scanner and record said data regarding the
intensity of said fluorescent light.
[*20] 20. The method of claim 14, wherein an open-ended hollow elongated
member having a reflective interior surface is used to diffuse said filtered
light.
[*21] 21. The method of claim 14, wherein said light measuring device is a
photomultiplier tube.
[*22] 22. The method of claim 14, wherein a beam expander is used to expand
said beam of light.
[*23] 23. A method for measuring the excited fluorescence of an object
comprising;
binding a specific dye to said object;
securing such object in a fixed position;
generating a coherent beam of light of specified wavelength within the range
of 350 to 540 nanometers;
expanding said beam of light;
focusing said expanded beam of light;
scanning such fixed object with said focused beam of light in a preprogrammed
three dimensional pattern and at a raster rate of 20 Hz while maintaining the
diameter of the light beam impinging upon said object at between 1 to 20
microns;
collecting the fluorescent light emitted from said object and attenuating all
other light by means of a biased cut optical fiber faceplate;
filtering said fluorescent light by means of a neutral density filter;
diffusing said filtered light in an open-ended hollow elongated member having
a reflective interior surface;
148
measuring said diffused light in a photomultiplier tube;
sequentially recording data regarding the intensity of said fluorescent light
as a function of the location of said scanning beam of light on said object; and
producing a visual image of at least a portion of said object based upon said
sequentially recorded data of the intensity of said fluorescent light as a
function of the location of said scanning beam of light.
[*24] 24. The method of claim 23, wherein a laser is used to generate said
coherent beam of light.
[*25] 25. The method of claim 23, wherein a programmable computer is used
to control the movement of said scanner and record said data regarding the
149
PAGE 29
Xxx. No. 4877966, *25
intensity of said fluorescent light.
[*26] 26. The method of claim 23, wherein a beam expander is used to expand
said beam of light.
150
FIGURES TO PATENT NO. 4,877,966
151
[FIG. 1 is a block diagram of a scanning device according to the present
invention.]
152
[FIG. 2 is an exemplary view of a scanning pattern of a translucent object with
a scanning device according to the present invention.]
153
[FIG. 3 is a block diagram of the electronic interface used to control the
various components of scanning device according to the present invention.]
154
[FIG. 4 is a block diagram showing an alternate embodiment of the fiber optic
bundle as used with a scanning device according to the present invention.]
155
[FIG. 5 is a flowchart representing the scanning program by which the present
invention operates.]
156
[FIG. 6a is an exemplary view of a biased cut optical fiber faceplate.]
157
[FIG. 6b is an exemplary view of a biased cut tapered optical fiber faceplate.]
158
[FIG. 7 is a schematic showing an analogue to digital convertor board as used in
the present invention.]
159
[FIG. 8 is a schematic showing the clock circuitry as used in the present
invention.]
160
[FIG. 9 is a schematic showing the low-order Address but Interface circuitry as
used in the present invention.]
161
[FIG. 10A, B is a schematic showing the DMA/Versabus Interface circuitry as used
in the present invention.]
162
[FIG. 11 is a schematic showing the DMA data path circuitry as used in the
present invention.]
163
[FIG. 12 is a schematic showing the timer interface circuitry as used in the
present invention.]
164
[FIG. 13a is a schematic showing the 1.5 volt local voltage source as used in
the present invention.]
165
[FIG. 13b is a schematic showing the Power-On Clear circuitry as used in the
present invention.]
166
[FIG. 14A, B is a schematic showing the board select logic as used in the
present invention.]
167
Exhibit C3 to Xxxxxx-Xxxxx License Agreement
168
US PATENT 4,758,727
<=2> GET 1st DRAWING SHEET OF 14
Jul. 19, 1988
Method and apparatus for the measurement of low-level
laser-induced fluorescence
INVENTOR: Tomei, L. Xxxxx, Dublin, Ohio
Cornhill, Xxxx, Worthington, Ohio
Xxxxxxxxx, Jogikal, Columbus, Ohio
Xxxxxxxx, Xxxxxxx, Columbus, Ohio
ASSIGNEE-AT-ISSUE: Ohio State University Research Foundation, Columbus, Ohio
(02)
APPL-N0: 828,694
FILED: Feb. 12, 0000
XXX-XX: [4] X00X 0#00
XX-XX: 250#458.1; 250#461.2
CL: 250
SEARCH-FLD: 250#458.1, 459.1, 461.2, 461.1; 350#96.27, 527
REF-CITED:
U.S. PATENT DOCUMENTS
3,450,455 6/1969 Xxxxxx
3,516,746 6/1970 Shibata et al.
3,981,590 9/1976 Xxxxxxx
4,107,534 8/1978 Piltingsrud 250#368
4,523,799 6/1985 Delhaye et al. 350#527
FOREIGN PATENT DOCUMENTS
0123942 11/1984 European Patent Organization 250#327.2
0155813 9/1985 European Patent Organization 250#368
169
OTHER PUBLICATIONS
Bussini et al, "A Silicon Rubber Scint. Comp. . . . for Rad. Det.", Nucl. Inst.
& Methods 107, #2, (1973) p. 333.
Xxxxxx, F., "Mural T.V. Display using Fiber Optics", RCA Technical Notes (RCA TN
No.:188); (1958).
Xxxx et al, "Vidicon Microscope for Count. Fluorescent Particles", Rev. of
Scient. Instru., vol. 42, #4, (1971) p. 508.
"The FACS Analyzer Fluorescence and Volume Cell Analysis" brochure, Becton and
Xxxxxxxxx FACS Systems, 1982.
"The New FACS 400 Series, FACS 440, FACS 420, FACS 400, Fluorescence Activated
Cell Sorting" brochure, Becton Xxxxxxxxx FACS Systems, 1981.
170
PAGE 31
Xxx. No. 4758727, *
"Photodigitizing with PDS Microdensitometer Systems" brochure, Xxxxxx-Xxxxx not
dated.
"Microdensitometer 3CS" brochure, Xxxxx-Xxxxx, not dated.
"Microdensitometer 6" brochure, Xxxxx-Xxxxx, not dated.
"EIKONIX Registered TM Applied Research Software and Product Development
Feasibility and Impact Studies Automated Instruments" brochure, EIKONIX
Registered TM Corp., not dated.
"ACAS 470 Workstation" Brochure, Meridian Instruments, Inc., not dated.
PRIM-EXMR: Xxxxxx, Xxxxxx X.
ASST-EXMR: Xxxxx, Xxxxxxx
LEGAL-REP: Emch, Schaffer, Xxxxxx & Xxxxxxxx Co.
ABST:
A method and apparatus for measuring low-level laser-induced fluorescence are
disclosed. A laser is used to produce a coherent beam of light which is
sequentially passed through a beam expander, iris diaphragm, focusing lens and
three-dimensional scanner before being focused onto a rigidly mounted target. A
computer is used to predeterminately control the pattern and the rate at which
the scanner passes the beam of light over the target. The light transmitted onto
the target induces fluorescent light in the target. The fluorescent light is
sequentially gathered by a biased cut optical fiber member and directed into a
photomultiplier tube where the intensity of the fluorescent light is measured.
The intensity data is then digitized and recorded by the computer as a function
of the coordinates of each preprogrammed point location of the beam impinging
upon the target. This data is used to produce an image of all or a portion of
the target on a visual monitor. In addition, the data can be recalled and used
for further analysis of the target.
NO-OF-CLAIMS: 26
EXMPL-CLAIM: <=8> 1
NO-OF-FIGURES: 18
NO-DRWNG-PP: 14
171
SUM:
BACKGROUND OF THE INVENTION
The present invention relates a method and apparatus for the measurement of
low-level laser-induced fluorescence in the field of cytofluorometry.
Cytofluorometry has been greatly enhanced with the development of fluorescence
dyes that are specific for deoxyribonucleic acid (DNA). These dyes permit the
determination of the ratios of total DNA present in a cell population and, since
the total amount of DNA doubles as a cell progresses through its proliferation
cycle, the distribution of cell cycle positions that existed in a cell
population is easily determined statistically. This is based upon the fact that,
within necessary tolerances, the amount of dye bound to a cell is directly
proportional to the amount of DNA present.
172
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Xxx. No. 4758727, *
Fluorescence is the emission of light whose wavelength is different from that
used to induce or excite the molecule of dye. Therefore, common fluorescence
techniques require that the dye bind specifically to some component which is to
be measured such as DNA. Such specificity is obtained by structurally modifying
the dye molecule or coupling a dye molecule to another molecule that has the
required specificity of binding. Another technique which utilizes fluorescence
emission is based upon other properties of some dye molecules. When these
molecules are in an aqueous environment, their fluorescence characteristics are
distinctly different from that obtained in a hydrophobic environment. Since all
biological membranes have hydrophobic regions, the amount and kind of
fluorescence obtained following staining is related to the structural state of
that membrane.
Several analytical techniques have been developed which exploit these various
properties of fluorescent dyes. These techniques include photo-bleaching,
fluorescence quenching, and shifts in fluorescence emission spectra. Problems
are continuously encountered with these various techniques in that total
fluorescence per cell, following any general technique with any particular dye,
varies markedly and the variation is not quantitated for the expressed purpose
of defining the degree or nature of cell cooperativity in a coordinating,
interacting, cell mass.
Another fluorescence technique, stereological computer assisted
cytofluorometry (SCAC) provides for the measurement of laser-induced cellular
fluorescence in a cell or tissue mass (e.g. monolayer cell cultures or tissue
sections). Dependent upon the nature of the fluorescent dye employed, the
cellular response-density distribution profile will provide data of profound
theoretical as well as practical significance. SCAC provides a technique for the
quantitation of cell behavior and responses to drugs within the context of the
cell mass or tissue. This has led to the development of new pharmacological
parameters and is expected to lead to more refined and sophisticated parameters
with which to study drug actions in the fields of cancer diagnosis,
chemoprevention and chemotherapy, environmental and forensic toxicology, as well
as basic biological sciences.
Currently there are two basic apparatuses for performing cytofluorometric
studies. A fluorescence microscope such as the FACS TM series analyzer
manufactured by Becton Xxxxxxxxx provides observation of cells contained in a
culture dish. The fluorescence microscope is highly accurate when analyzing a
small area but cannot measure cell groupings larger than a culture dish without
destroying the spatial relationship of the cells. The second apparatus, a Flow
173
Cytometer is designed to provide observation for a large number of cells, but
the cells must be in suspension, thereby eliminating any possibility of
obtaining spatial data. The CYTOFLUOROGRAF system from the Ortho Instruments
Corporation is an example of a flow cytometer. The flow cytometer provides
highly accurate information on the frequency distribution of fluorescence
intensity in a randomly dispersed cell populations. However, no information is
provided regarding the spatial relationship that may exist between cells in the
tissue, tumor or culture prior to dispersal and staining.
Interpretation of frequency distributions of fluorescence intensity is
seriously hampered by the fact that the response of the original cell population
is rarely spatially homogenous. Heterogeneity of cell identity, morphology and
drug responsiveness is commonly observed but not considered in current
cytofluorometric analytic techniques. However, population heterogeneity is
174
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Xxx. No. 4758727, *
regarded by biologists as being an inherent quality of coordinating cell
populations found in all animal tissues, tumors, primary cell cultures, and in
rudimentary form, laboratory cell lines. The present invention, method and
apparation, provides the ability to observe large areas of tissue, while
maintaining full spatial relationships without the need for any special
preparation.
SUMMARY OF THE INVENTION
The present invention relates a method and apparatus capable of rapid
wide-field scanning of low-level laser induced fluorescence of tissue sections,
cell cultures, and other biological materials, while maintaining high spatial
resolution. The method and apparatus of the present invention measures off-axis
fluorescence. The fluorescence measurements permits full digitalization of
images with 16 bit precision for 2-4 x 10<6> pixels. The target areas scanned
are greater than 25 cm<2> with a resolution of 5-10 mu m and the scanning time
is between 1 and 6 seconds dependent upon computer processing and storage
limitations. The SCAC methodology, provides for the acquisition of information
from a large number of individual cells as does flow cytofluorometry. However,
in addition to individual cell responses, the present invention will provide
highly accurate information regarding the spatial distribution of those cells
within the total population. This is of particular importance in the areas of
tumor biology, pathology and early detection of abnormal cells in tissues and
organs. The SCAC design of the present invention provides for analysis of large
cell numbers in vitro with high spatial precision.
The apparatus of the present invention incorporates an optical fiber taper
for high efficiency light gathering and transmission to a highly sensitive
detector. The properties of the fibers can be exploited to shift the detector
off-axis. This apparatus of the present invention makes use of two fiber
properties: tapered fiber transmission and biased cut deflection. Further,
extra-mural absorption material are added to provide further attenuation of
off-axis incident laser light. The method and apparatus of the present invention
will be further understood by the following description of the preferred
embodiment with reference to the following figures.
DRWDESC:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a block diagram of a scanning device according to the present
invention.
175
FIG. 2 is an exemplary view of a scanning pattern of a translucent object
with a scanning device according to the present invention.
FIG. 3 is a block diagram of the electronic interface used to control the
various components of a scanning device according to the present invention.
FIG. 4 is a block diagram showing an alternate embodiment of the fiber optic
bundle as used with a scanning device according to the present invention.
FIG. 5 is a flowchart representing the scanning program by which the present
invention operates.
FIG. 6a is an exemplary view of a biased cut optical fiber faceplate.
176
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Xxx. No. 4758727, *
FIG. 6b is an exemplary view of a biased cut tapered optical fiber faceplate.
FIG. 7 is a schematic showing an analogue to digital convertor board as used
in the present invention.
FIG. 8 is a schematic showing the clock circuitry as used in the present
invention.
FIG. 9 is a schematic showing the low-order Address bus Interface circuitry
as used in the present invention.
FIG. 10 is a schematic showing the DMA/Versabus Interface circuitry as used
in the present invention.
FIG. 11 is a schematic showing the DMA data path circuitry as used in the
present invention.
FIG. 12 is a schematic showing the timer interface circuitry as used in the
present invention.
FIG. 13a is a schematic showing the 1.5 Volt local voltage source as used in
the present invention.
FIG. 13b is a schematic showing the Power-On Clear circuitry as used in the
present invention.
FIG. 14 is a schematic showing the board select logic as used in the present
invention.
DETDESC:
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention relates to a method and apparatus for the off-axis
detection of laser-induced fluorescence. Referring to FIG. 1, there is shown a
block diagram of the components of a scanning device 10 according to the present
invention. The scanning device 10 uses coherent light to scan a object or target
12. In the preferred embodiment, the components of the device 10 include; a
tuneable laser 14, a beam expander 16, an iris diaphragm 18, a focusing lens 20,
a three-dimensional scanner 22, a target mount 24, a fiber optic faceplate
having a bias cut 26, a diffusion member 28, a photomultiplier tube 30, a
177
programmable computer with memory 32 and a visual monitor 34. Together these
components provide for rapid scanning of translucent targets to yield high
resolution, wide-field fluorescence analyses of a large number of cells in
vitro, with high spatial precision.
The means for generating the coherent beam of light is a laser 14. It is
desirable that the laser 14, shown in FIG. 1 emit a coherent polarized beam of
light 15 having a wavelength in the range of 350 to 540 nanometers. The light
beam 15 exiting the laser 14 is approximately 1 millimeter in width, which is
too small to be used effectively by the reflecting mirrors within the scanner
22. Therefore, the present invention provides a beam expander 16 which receives
the laser beam 15 and enlarges the size of the beam 15 to ensure utilization of
the greatest possible surface area of the reflecting mirrors within the scanner
22. The preferred beam expander 16, as shown in FIG. 1, is a Buschnell
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variable beam expander having a magnification power range of 9 x to 30 x . In
the preferred embodiment, the beam expander 16 is adjustable to expand the beam
size through a range from 1 millimeter to 10 millimeters.
As an alternative to the use of a variable beam expander, a single power beam
expander may also be used with Applicant's invention. In such cases an iris
diaphragm 18 is included to receive the expanded beam 15 from the single power
beam expander. The iris diaphragm 18, also known as a field iris, is intended to
vary the diameter of the beam 15 input to the focusing lens 20 and the scanner
22, thus varying the spot size on the target 12. Consequently, the iris
diaphragm 18 is used to more finely tune the size of the beam of light 15.
Referring now to FIG. 1, the beam of light 15 exiting the beam expander 16 or
iris diaphragm 18 is passed through the focusing lens 20 which focuses the beam
of light 15 on the target 12. Together, the beam expander 16/iris diaphragm 18
and focusing lens 20 permit the beam of light to be focused to a spot of
predetermined dimension at the target plane of the translucent target 12.
In the preferred embodiment, the light received by the focusing lens 20 is a
gaussian columnated coherent beam of light. Using this assumption, the final
spot size upon the target 12 is diffraction limited and governed by the
following equation:
d = 4F lambda / pi D, (1)
wherein
d = spot size
F = focal length of focusing lens
lambda = wavelength of the light
D = aperture of input beam to focusing lens
According to equation (1), the proper selection of the focal length (F) of
the focusing lens 20, the wavelength ( lambda ) of the coherent light beam 15,
and the aperture (D) of the input beam to the focusing lens 20, will allow for
the size of the beam spot (d) on the target 12 to be varied as desired. The
smallest available spot size is approximately one micron. Thus, the present
invention is capable of scanning at a variety of intervals with the smallest
179
being a one micron interval.
The focused beam of light 15 exiting the focusing lens 20 is received by the
three-dimensional scanner 22 which operates to control the passing of the light
beam 15 over the target 12. The scanner 22 as shown in FIG. 1 is manufactured by
General Scanning, Inc. and uses computer controlled mirrors to pass the focused
beam of light 15 back and forth across the target 12. The scanner 22 is capable
of scanning an area of any size, up to 20 millimeters by 40 millimeters, while
operating at a raster rate of up to 20 Hz or a vector rate of up to 280 mm/sec.
Computer controlled drive motors cause the mirrors of the scanner 22 to move
such that the scanner 22 has accurate X and Y coordinate scanning capabilities
with a simultaneous Z coordinate correction to yield a beam of light 15 with a
flat field of focus. If desired, the Z coordinate of the scanner 22 can be
programmed to coordinate with an uneven topographical field of focus on the
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Xxx. No. 4758727, *
target 12.
The scanner 22 passes the focused coherent beam of light to the target 12,
thereby inducing fluorescence in a predetermined pattern and at a predetermined
rate set by the computer 32 so that data is obtained as to the intensity of the
fluorescence at differing points on the target 12. At precise predetermined
points during the scanning process, the intensity of the light is measured by
the photomultiplier tube 30 and recorded by the computer 32 as a function of
location of the beam 15 on the target 12. By taking successive point
measurements of the intensity of the fluorescence and relating them back to the
location data in the computer 32, the data is used to analyze the target 12 or
recreate a visual image of the target 12 on a visual monitor 34.
The target 12 is scanned over a very large series of points using a back and
forth pattern as shown in FIG. 2. The X, Y coordinates and the rate of scanning
are predetermined and programmed through the computer 32. The scanning program
is individually drawn to the specifics of the application. The flowchart for the
scanning program is shown in FIG. 5. At each scanning point, the fluorescence
intensity is measured and recorded as a function of the X, Y coordinates of the
beam. For example, the scanner 22 would be programmed to scan the target 12, as
shown in FIG. 2, in a sequential pattern over the following (X,Y) coordinates:
(1,1), (1,2), (1,3), (1,4), (2,4), (2,3), (2,2), (2,1), (3,1), (3,2), (3,3),
(3,4), (4,4), (4,3), (4,2) and (4,1). The intensity of the fluorescence being
excited at each of the scanning points is measured by the photomultiplier tube
30 in terms of analogue data. The analogue data for each scanning point is then
digitized by the computer 32 and recorded as a function of the X, Y coordinates
of that particular scanning point.
The present invention utilizes a three-dimensional scanner, such as that
manufactured by General Scanning, Inc. The use of such a scanner provides in
that the target mount 24 is stationary, thereby eliminating the wobble and
jitter experienced with moving target mounts. Due to the minute size of the
focused beam spot on the target 12 and the large amount of point data being
taken, it is important that the target 12 be rigidly mounted in a fixed position
on the target mount 24 to maintain the proper focusing, depth of field, and spot
size.
Positioned directly below the target mount 24 and thus below the target 12
itself is a fiber optic faceplate 26 which is used in conjunction with a
diffusion member 28 to increase the efficiency of the highly sensitive
photomultiplier tube 30. The sensing surfaces of photomultiplier tubes are
181
characteristically nonuniform. The fluorescent light transmitted by the target
12 is minute in the form of a narrow columnated beam. If this beam were received
by the photomultiplier tube 30, variations in the recorded intensity of the beam
would occur as a result of variations in the location at which the narrow
columnated light beam strikes the sensing surface of the photomultiplier tube
30. Therefore, the present invention utilizes the fiber optic faceplate 26 to
gather as much light as possible and the diffusion member 28 spreads the light
beam broadly as possible to achieve full use of the photomultiplier tube 30.
One of two types of fiber optic faceplates 26 may be used with the present
invention, depending upon the size of the window of the photomultiplier tube 30.
When the window of the photomultiplier tube 30 is comparable in size to the
target 12, then a flat disk-type faceplate, as shown in FIGS. 1 and 2, is used.
When the scanning area of the target 12 is larger than the window of the
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photomultiplier tube 30, a tapered or frustoconical shaped fiber optic faceplate
26A, such as shown in FIG. 4, is used. The use of such fiber optic faceplates
presents a marked improvement over prior art systems in which lenses are relied
upon to gather and direct the light beam. The light gathering or flux-carrying
capacity of the optical fibers within the taper 26A as used with the present
invention is 10 to 70 times higher than that of standard optical lenses. The
relative increase in light gathering capacity is numerically equal to the ratio
of the squares of the numerical apertures. The effective numerical aperture of a
typical lens capable of imaging a target area of at least several square
millimeters is 0.10 to 0.20 as compared with optical fibers which have nominal
numerical aperture values of greater than 0.60. Therefore, the use of optical
fibers in conjunction with highly sensitive photomultiplier tubes 30 produces a
marked advantage over instrument designs incorporating optical lens systems
and/or solid-state light detectors.
The fiber optic faceplate 26 is biased cut at a angle alpha , preferably 30o.
Referring to FIG. 6, the bias cut on the optical fiber faceplate 26 is shown as
angle alpha and the acceptance angle of the fiber optic faceplate 26 is
deflected by angle beta . This provides a total deflection by angle beta or
shift of the acceptance angle theta which comprises the acceptance cone of the
fiber optic bundle. If laser light is shown onto a fiber outside of the
acceptance cone, the amount of light transmitted along the length of the fiber
will be severely limited. Thus, the present invention provides a fiber optic
faceplate 26 having a bias cut such that light hitting perpendicular to the face
of the fibers will not be in the acceptance cone. In this manner, a light source
hitting perpendicular to the surface will be attenuated while excited
fluorescence or scattered light may enter the acceptance cone. The bias cut
fiber optic system of the present invention is capable of gathering 30% of the
excited fluorescence from the target being scanned, as compared with current
technology in which only 2% of the excited fluorescent light is gathered.
The optical fibers in the faceplate 26 also function as the first stage for
light diffusion. Optical fibers will gather light from all angles within the
acceptance cone. Upon exit from the faceplate 26 this light is rotated about a
solid angle through which the intensity is uniformly distributed. This optic
fiber property thus enhances the light scattering efficiency of the diffusion
member 28.
Further attenuation of the light is accomplished by insertion of neutral
density filters 27 between the fiber optic faceplate 26 and the diffusion member
28. In the preferred embodiment, a 620 nanometer filter is incorporated in order
183
to eliminate the normal fluorescent room lighting, thus permitting operation of
the instrument without extensive light protection. Further, a barrier filter
(not shown) may be incorporated at this point to absorb light at the excitation
wavelength while transmitting light at the fluorescent wavelength.
The diffusion member 28, shown in FIG. 1, is an open-ended hollow elongated
member with a reflective interior surface. Such a member can be constructed of
an open-end box using mirrored glass for the interior surface. Alternatively,
milk glass may be used as the diffusion member 28. The primary functions of the
diffusion member 28 are two-fold. First, the diffusion member 28 blocks out any
remaining ambient light which will affect the intensity readings of the
photomultiplier tube 30. Secondly, the diffusion member 28 allows the
transmitted light to diffuse within its confines to more fully utilize the
window of the photomultiplier tube 30 and thus yield more accurate results.
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Xxx. No. 4758727, *
After the fluorescent light transmitted from the target 12 is directed to the
photomultiplier tube 30 by the fiber optic faceplate 26 and diffusion member 28,
the photomultiplier tube 30 detects and measures the fluorescence levels. The
photomultiplier tube 30 is in turn linked to the computer 32 so that data
obtained by the photomultiplier tube 30 regarding the intensity of the light is
fed into the computer 32.
Once the device 10 is activated and scanning of the target 12 begins, the
output of the photomultiplier tube 30 is sampled by the computer 32 as often as
desired. The electrical signals relating the intensity of the fluorescent light
are converted from analogue to digital values by the computer 32 for storage
purposes. Using the preprogrammed position data and the stored intensity values,
the computer 32 can provide a realtime pictorial reconstruction of the target 12
on a visual monitor 34. The capacity of the computer 32 to rapidly store the
highly detailed data being viewed makes it possible to depict the entire area of
the target 12 on the visual monitor as well as focus on particular points of
interest by providing enlarged viewing of select portions of the target 12.
Referring now to FIG. 3, an interface is provided to collect the analogue
intensity data output of the photomultiplier tube 30 and convert the data into
digital form for the versabus-based computer 32. The interface 33 communicates
with the computer 32 through direct memory access (DMA) with a minimum
instantaneous transfer rate of 100,000 picture elements (pixels) per second. The
interface 33 of the present invention is composed of five main subsystems
including: an analogue-to-digital (A/D) converter 36 as shown in FIG. 7; a set
of buffers and latches 38; bus interface logic 40 as shown in FIGS. 9, 10; a DMA
controller 42 as shown in FIG. 11; and a system timing controller 44 as shown in
FIG. 12. The DMA controller 42 and system timing controller 44 are used to
properly operate the A/D converter 36, the set of buffers and latches 38 and the
bus interface logic 40.
Referring now to FIG. 7, the A/D converter 36 transforms the image data
signals which are received from the photomultiplier tube 30 into a digital form
which is understandable by the host computer 32. The buffers change the voltage
of the signals to a level which is compatible with the host computer 32 while
the latches store the transformed data until it is efficient for the computer 32
to accept them.
Referring now to FIG. 11, the DMA controller 42 and the bus interface logic
40 of FIGS. 9 and 10 store and control information about the flow of the data to
the computer 32 so that the computer 32 receives the data only when it is able
185
to accept the information. The system timing controller 44 of FIG. 12 ensures
that the proper number of pixels are placed into the memory 46 of the computer
32. In addition, the system timing controller 44 generates all the needed
signals for the A/D converter 36.
Since the A/D converter 36 outputs emitter coupled logic (ECL) compatible
signals, level shifters are required to provide transistor-transistor logic
(TTL) signals. These signals are tied directly to a combination bus buffer/latch
chip. The conversion timing is derived from a 1 MHz crystal feeding an AM9513
timing controller. It should be noted that higher frequency crystals may be
used. The timing controller is started by means of an enable convert signal
which can be generated by the host computer 32 under user control, or by a
signal external to the board. The timing controller 44 generates the convert and
latch signal, and also terminates its count automatically. The controller is
completely software
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Xxx. No. 4758727, *
programmable.
The bus interface logic permits the use of both a positive true logic and
negative true logic CPU bus. The bus also allows for a choice of an 8 or 16 MHz
CPU clock speed. The board is memory-mapped, and may reside on any 256 byte
boundary in memory.
The DMA controller circuit is designed to provide for both cycle steal and
burst modes, under software programmability. This feature allows for increased
efficiency at high data rates. Regardless of the mode chosen, the eight most
significant bits are latched, and the next sample is latched in the same manner.
At this point, the DMA transfer will proceed in the specified mode. It is
important to note that by first latching 16 bits and then requesting the bus,
the bus bandwidth used in cycle steal mode is cut to one half of what it would
otherwise have been.
The remainder of the circuitry includes a 1.5 volt local power supply 48
(FIG. 13a), as required by the DMA control circuitry, and a dual speed
power-on-clear circuit 50 (FIG. 13b) which included to ensure the circuit powers
up in a predictable and stable manner.
With the circuitry in place, the device 10 is used to scan the object 12. The
excited fluorescent intensity is determined using the highly efficient optical
fiber faceplate 26 and the sensitive photomultiplier tube 30 without respect to
the precise position of the laser beam on the target object. Such position is
alternatively determined by the computer 32 which controls the three-dimensional
scanner 22. The computer 32 then combines the X, Y coordinate spatial
information with the intensity information to yield a high resolution wide-field
image of the object 12.
Having thus described the invention in detail, it should be understood that
various modifications and changes can be made in the apparatus without departing
from the scope and content of the following claims.
CLAIMS: What we claim is:
[*1] 1. An apparatus for measuring the excited fluorescence of an object
comprising:
means for generating a coherent beam of light;
187
a three dimensional scanner having means for securing such object in a fixed
position, said scanner receiving and passing said coherent beam of light onto
such fixed object in a desired pattern and at a desired rate, said scanner
having X, Y coordinate scanning capability with simultaneous Z coordinate
correction to cause said beam to have a topographical field of focus in the
place of said object;
means for controlling the pattern and rate at which said beam of light is
passed onto said object;
an optical fiber member for gathering and directing the fluorescent light
induced from said object by said beam of light and for attenuating any direct
light received by said object;
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Xxx. No. 4758727, *1
means for collecting and measuring the intensity of the fluorescent light
received from said object; and,
means for sequentially recording data regarding the intensity of the
fluorescent light received from said object as a function of the location of
said scanning beam of light on said object by said scanner.
[*2] 2. The apparatus of claim 1 further including a means for filtering
said fluorescent light emanating from said optical fiber member.
[*3] 3. The apparatus of claim 1 further including a means for diffusing
said fluorescent light emanating from said optical fiber member.
[*4] 4. The apparatus of claim 1, further including means for producing a
visual image of at least a portion of said object based upon said sequentially
recorded data of the intensity of said transmitted light as a function of the
location of said scanning beam of light on said object.
[*5] 5. The apparatus of claim 1, which further includes a focusing lens
interposed between said means for generating a coherent beam of light and said
three dimensional scanner.
[*6] 6. The apparatus of claim 5, which further includes a beam expander
interposed between said means for generating a coherent beam of light and said
focusing lens.
[*7] 7. The apparatus of claim 6, which further includes an iris diaphragm
interposed between said beam expander and said focusing lens.
[*8] 8. The apparatus of claim 1, wherein said means for diffusing said
transmitted light comprises an open-ended hollow elongated member having a
reflective interior surface.
[*9] 9. The apparatus of claim 1, wherein said means for collecting and
measuring the intensity of the light transmitted through said translucent object
comprises a photomultiplier tube.
[*10] 10. The apparatus of claim 1, wherein said means for predeterminately
controlling the pattern and rate at which said beam of light is passed over said
object is a programmable computer.
189
[*11] 11. The apparatus of claim 1, wherein said means for sequentially
recording data is a programmable computer.
[*12] 12. An apparatus for measuring the excited fluorescence of an object
having a binding fluorescent dye application comprising:
means for generating a coherent beam of light;
means for expanding said coherent beam of light;
means for focusing said expanded beam of light;
a three dimensional scanner having means for securing such object in a fixed
position, said scanner receiving and passing said focused beam of light over
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Xxx. No. 4758727, *12
such fixed object in a selected pattern and at a selected rate, said scanner
having X, Y coordinate scanning capability with simultaneous Z coordinate
correction to cause said focused beam of light to have a topographical field of
focus in the plane of said object;
means for controlling the pattern and rate at which said beam of light is
passed through said translucent object;
a biased cut optical fiber faceplate for receiving and directing the
fluorescent light transmitted from said object;
means for filtering said fluorescent light emanating from said fiber
faceplate;
means for diffusing said filtered light;
means for collecting and measuring the intensity of the fluorescent light
transmitted through said object; and
means for sequentially recording data regarding the intensity of the
fluorescent light transmitted from said object as a function of the location of
said scanning beam of light on said object.
[*13] 13. The apparatus of claim 12, which further includes means for
producing a visual image of at least a portion of said object based upon said
sequentially recorded data as a function of the location of the fluorescent
light transmitted from said object.
[*14] 14. The apparatus of claim 12, which further includes an iris diaphragm
interposed between said means for expanding said beam of light and said means
for focusing said expanded beam of light.
[*15] 15. The apparatus of claim 12, wherein said means for generating a
coherent beam of light is a laser.
[*16] 16. The apparatus of claim 12, wherein said means for expanding said
beam of light is a variable beam expander.
[*17] 17. The apparatus of claim 12, wherein said means for controlling the
pattern and rate at which said beam of light is passed over said object is a
programmable computer.
191
[*18] 18. The apparatus of claim 12, wherein said means for diffusing said
fluorescent light is an open-ended hollow elongated member having a reflective
interior surface.
[*19] 19. The apparatus of claim 12, wherein said means for collecting and
measuring the intensity of the fluorescent light transmitted from said
translucent object is a photomultiplier tube.
[*20] 20. The apparatus of claim 12, wherein the means for sequentially
recording data is a programmable computer.
[*21] 21. An apparatus for measuring the excited fluorescence of an object
having a finding fluorescent dye application comprising;
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Xxx. No. 4758727, *21
a laser for generating a coherent beam of light at a specified wavelength
with the range of 350 to 540 nanometers;
a variable beam expander for expanding said coherent beam of light;
a focusing lens to focus said expanded beam of light;
a three dimensional scanner having means for securing such object in a fixed
position, said scanner receiving and passing said focused beam of light over
such fixed object in a selected pattern and at a selected rate, said scanner
having X, Y coordinate scanning capability with simultaneous Z coordinate
correction to cause said focused beam of light to have a topographical field of
focus in the plane of said object;
means for controlling the pattern and rate at which said focused beam of
light is passed over said object;
a biased cut optical fiber faceplate for receiving and directing the
fluorescent light transmitted from said object;
filter means for receiving said fluorescent light from said fiber optic
bundle and removing unwanted light input;
diffusion means for diffusing said filtered light;
a photomultiplier for measuring the intensity of the fluorescent light
transmitted and received from said diffusion means; and,
means for sequentially recording data regarding the intensity of the
fluorescent light transmitted from said object as a function of the location of
said scanning beam of light on said object.
[*22] 22. The apparatus of claim 21, wherein said means for controlling the
pattern and the rate at which said focused beam of light is passed over said
object is a programmable computer.
[*23] 23. The apparatus of claim 21, wherein said means for sequentially
recording data is a programmable computer.
[*24] 24. The apparatus of claim 21, wherein said diffusion means is an
open-ended hollow elongated member having a reflective interior surface.
193
[*25] 25. The apparatus of claim 21, which further includes an iris diaphragm
interposed between said beam expander and said focusing lens.
[*26] 26. The apparatus of claim 21, which further includes means for
producing a visual image of at least a portion of said object based upon said
sequentially recorded data as a function of the location of the fluorescent
light transmitted from said object.
194
FIGURES TO PATENT NO. 4,758,727
195
[FIG. 1 is a block diagram of a scanning device according to the present
invention.]
196
[FIG. 2 is an exemplary view of a scanning pattern of a translucent object with
a scanning device according to the present invention.]
197
[FIG. 3 is a block diagram of the electronic interface used to control the
various components of scanning device according to the present invention.]
198
[FIG. 4 is a block diagram showing an alternate embodiment of the fiber optic
bundle as used with a scanning device according to the present invention.]
199
[FIG. 5 is a flowchart representing the scanning program by which the present
invention operates.]
200
[FIG. 6a is an exemplary view of a biased cut optical fiber faceplate.]
201
[FIG. 6b is an exemplary view of a biased cut tapered optical fiber faceplate.]
202
[FIG. 7 is a schematic showing an analogue to digital convertor board as used in
the present invention.]
203
[FIG. 8 is a schematic showing the clock circuitry as used in the present
invention.]
204
[FIG. 9 is a schematic showing the low-order Address but Interface circuitry as
used in the present invention.]
205
[FIG. 10 is a schematic showing the DMA/Versabus Interface circuitry as used in
the present invention.]
206
[FIG. 11 is a schematic showing the DMA data path circuitry as used in the
present invention.]
207
[FIG. 12 is a schematic showing the timer interface circuitry as used in the
present invention.]
208
[FIG. 13a is a schematic showing the 1.5 volt local voltage source as used in
the present invention.]
209
[FIG. 13b is a schematic showing the Power-On Clear circuitry as used in the
present invention.]
210
[FIG. 14 is a schematic showing the board select logic as used in the
present invention.]
211
CONFIDENTIAL TREATMENT REQUESTED
LXR BIOTECHNOLOGY INC.
LOCATED AT 0000 XXXXXX XXX XXXXX
XXXXXXXX, XXXXXXXXXX 00000
Exhibit C4 to Xxxxxx-Xxxxx License Agreement
212
**CONFIDENTIAL TREATMENT REQUESTED
MULTI-CHANNEL ACQUISITION USING INTEGRATING SPHERE
** [ ]
213
CONFIDENTIAL TREATMENT REQUESTED
LXR BIOTECHNOLOGY INC.
LOCATED AT 0000 XXXXXX XXX XXXXX
XXXXXXXX, XXXXXXXXXX 00000
Exhibit C5 to Xxxxxx-Xxxxx License Agreement
214
**CONFIDENTIAL TREATMENT REQUESTED
WIDE ANGLE SCATTERING DETECTOR
** [ ]
215
CONFIDENTIAL TREATMENT REQUESTED
LXR BIOTECHNOLOGY INC.
LOCATED AT 0000 XXXXXX XXX XXXXX
XXXXXXXX, XXXXXXXXXX 00000
Exhibit C6 to Xxxxxx-Xxxxx License Agreement
216
** CONFIDENTIAL TREATMENT REQUESTED
[**]
