Common use of Device Operation Clause in Contracts

Device Operation. All fluids were injected into the microfluidic device by loading into individual syringes (Gastight, Xxxxxxxx) driven by syringe pumps (PHD 22/2000, Harvard Apparatus). Protein solutions or bead suspensions were mixed with a solu- tion of substrate (500 μM fluorescein-di-β-D-galactopyranoside, FDG, Invitrogen) in PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.4, Ambion) containing 0.1% v/v Tween-20 (Sigma) in the microfluidic device prior to droplet generation at the flow-focusing nozzle. Water droplets are formed in fluorinated oil (HFE-7500, Novec, 3M) previously mixed with a surfactant (5% w/w, Methods) to generate dro- plets stably and prevent their coalescence. × + Fluorescence Image Acquisition and Analysis. Fluorescence images were obtained using an inverted microscope (IX71, Olympus) operated in epifluorescence mode using a mercury lamp (U-H100HG, Olympus) as an excitation source. The micro- fluidic devices were illuminated, and the emitted light was collected using the same objective (UPLSAPO 40 2, Olympus); excitation light was passed through a neutral density filter (25% transmission, Olympus) and only during image acquisi- tion, in order to minimize photobleaching. Excitation light was spectrally filtered and separated from fluorescence emission using two mirror sets (excitation 475 ( 17 nm/emission 530 ( 22 nm, and excitation 559 ( 17 nm/emission 630 ( 35 nm, Thorlabs); green- and red-fluorescence micrographs were col- lected sequentially using a motorized filter cube (IX2-RFACA-1-5, Olympus) to alternate between the two colors. Images were acquired using an EMCCD camera (Xion , Andor Technologies) with exposure times of 0.1 and 1 s for red and green fluorescence, respectively. Image analysis was performed using custom soft- ware written in LabView, which calculated the fluorescence intensity of femtodroplets by integrating the brightness of all the component pixels of each droplet. × ∼ Measurement of Femtodroplet-Generation Frequencies. A high- precision optical setup was used to measure the frequency of droplet formation. Briefly, the 488 nm beam of a diode laser (Spectra-Physics) was directed to the back port of an inverted microscope (Eclipse TE2000-U, Nikon), where it was reflected by a dichroic mirror and focused 2 μm above the cover slide into the flow-focusing nozzle of the microfluidic device, using an oil- immersion objective (Apochromat 60 , NA 1.40, Nikon). Fluo- rescence was collected by the same objective and imaged onto a 70 μm pinhole (Melles Griot) to exclude out-of-focus light, forming a confocal detection volume of 0.1 fL. Green fluores- cence was filtered by a pair of long-pass and band-pass filters (540ALP and 535AF45, Omega Optical Filters) before being focused onto an avalanche photodiode (APD, SPCM-14, Xxxxxx- Xxxxx). The readout from the APD was coupled to a PC- implemented multichannel scalar (MCS) card and analyzed with custom-written software. Fluorescence was recorded as raw photon counts over a typical window of 50 ns. Femtodroplets were generated as described above, using 0.2 μM fluorescein in PBS buffer containing 0.1% v/v Tween-20 as the aqueous phase, and the frequency of droplet formation was calculated from the separation of the resulting fluorescence bursts in the MCS output. A fast camera (MIRO4, Vision Research) was used to visually confirm droplet generation and enable alignment of the confocal spot within the flow-focusing nozzle, while an extremely high speed camera (up to 1.0 Mfps, V1610, Vision Research) was used to capture movies of droplet generation (Supporting Movie 2). ARTICLE