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 objectiv...
Device Operation. The Xxxxxx Monitoring System is comprised of an ORCA designed and constructed monitoring instrument that measures flow speed, flow direction, water depth and water temperature. The Xxxxxx Monitoring system is integrated with a YSI EXO2 sonde with probes measuring ph/ORP, Conductivity/Temperature, Optical DO, Turbidity, total algae that includes chlorophyll and blue green algae sensors and fDOM. Additionally, the Xxxxxx is integrated with a NuLab nutrient monitoring system that monitors nitrate, nitrite and ortho-phosphate by utilizing wet chemistry which is not affected by bromides, water color changes and other contaminants that affect optical nutrient sensors. The integrated package is coupled to an ORCA designed and constructed communications system that reports to the ORCA designed database at 4-hour intervals. The complete system is solar powered and utilizes a battery backup for times when sunlight is not available. Each deployment site will be visited no less than once per month at which time YSI measurements will be verified using a YSI handheld sonde identical to the deployed instrument measuring salinity/conductivity/temperature, dissolved oxygen, pH/ORP, fDOM, total algae, and turbidity. The handheld sonde will be calibrated and verified with applicable portions of DEP SOPs FT 1000 (field testing general), FT 1100 (pH), FT 1300 (salinity), FT 1500 (DO) and FT 1900 (field continuous monitoring) prior to the site visit, and then verified after the monthly site visits. Verifications for pH and salinity will be conducted with standards that bracket the sample readings from the field. If post- sampling verifications do not meet acceptance criteria listed, associated sample data will be qualified with “J” (estimated). Any deployed instrument found out of calibration will be returned to the lab for calibration and check before redeployment. Calibration checks will be recorded in the ORCA database prior to and after recalibration. At each monthly site visit grab samples will be obtained by ORCA team members Xxxxx Xxxxx and Xxxxxx Xxxxxxx. Grab samples will be sent overnight to PACE Analytical Services, Inc. for comparability assessment of Nitrate/Nitrite, Orthophosphate and Chlorophyll a. At each monthly maintenance check, reagents for the NuLab nutrient monitoring device will be replenished and a grab sample will be obtained for verification.
