901303 Cytometer Technologies for Autonomous Platforms : Visit by Heidi Sosik on 9-11-2013 This page last changed on Sep 18, 2013 by oreilly. O'Reilly's notes on 10 am meeting. Attendees: Heidi Sosik, Denis Klimov, Tom O'Reilly Questions: Plans for small/low-power instrument? Can FCB/IFCB be deployed without power/data cable-to-shore? HS: Working on "low-relief" IFCBT for deployment on Wave Glider, AUV, other platform Current IFCB takes 30W power Particle size range overlap of FCB and IFCB? HS: Difficult; IFCB laser is optimized for microplankton - picoplankton requires higher-power laser. FCB is still original prototype (large) - no funding or plans to refine. CytoBuoy claims pico-micro capability. Could you replace sheath fluid with virtual core technology, ala SeaFlow (Need to add position-sensitive detector(s))? HS: Would be concerned about flow-cell cleanliness - core fluid keeps particles moving through flow cell. Very occassionally get dirt in cell - would this be a greater problem with SeaFlow/SeaLabel? Processing: HS: Several stages; detect particles (reject particle-free images regions). Edge detection, etc. Classification Now using "random forest" approach instead of SVM (2007): - rf requires less tuning than svm - svm requires complete characterized dataset for error characterization; rf doesn't - both methods yield comparable accuracy How long did the image-processing/classification procedures take per image? On what hardware? HS: particle detection/empty-image rejection executes in situ on instrument's "Atom" processor. Shore-side workflow in realtime, posted on MVCO website (http://ifcb-data.whoi.edu/) HS: Image processing is more computationally intensive than classification Klimov's notes from 10:00, 12:00, 16:00 meetings Principles of operation: - FlowCytobot / Imaging FlowCytobot sample path arranged in such a way as sample goes "down hill" due to plankton sinking. - Newer Imaging FlowCytobot (licensed to Mclane) is developed entirely at WHOI. McLane did some adaptations to simplify production. 8 inch diameter tube, 40 inch long. First unit built in August 2013. - 5ml sip, then 20 minutes analyzing. This time can be reduced by sipping less, say 1ml then 4 min analyzing, but the period can not be much shorter due to time overhead (pump start/ stop etc) and required statistics. - this design has emphasis on accurate statistics so flow and volume need to be precise, so syringe pump is used with defined volume. - Emphasis on "binning" to get particle statistics per "shot" and not as continuous measurement; so "binning" is ok here. - Also, accurate flow rate is required, due to separation of "trigger volume" and image acquisition volume within a sample cuvette, and fixed time delay between trigger and acquisition of the image. Image is acquired with 1 us light pulse from Xenon lamp. - Sheath fluid is preventing the contact between sample water and walls of the cuvette, minimizes chance of contamination and particles attachment to cuvette walls, very important feature. - Sheath fluid is recirculated by filtering sample + sheath by volumetric Fisher filter. Excess water is dumped into environment before filter. Filter lasts 6-8 month deployment easy. Use of Imaging FlowCytobot on a mobile platform: - Configuration of the sample path dictates "vertical" position of the instrument so it operation in horizontal orientation could be problematic. - For glider use, perhaps the Imaging FlowCytobot can be split into set of housings and then arrange them horizontally. - Suggestion: keep volume of those compartments relatively small (under several hundred mL) to avoid vehicle loss in case of flooding one compartment. Difference between FlowCytobot and Imaging FlowCytobot: - Particle ranges: FlowCytobot 1-15 um, Imaging FlowCytobot 5-200um - Cuvettes have different geometries: FlowCytobot has smaller cylindrical flow cavity for tighter light collimation of laser, and Imaging FlowCytobot has "50 um sheet" of fluid optimized for imaging and larger laser beam for triggering - Light sources are optimized differently for smaller circular path, or wider flat path

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