901303 Cytometer Technologies for Autonomous Platforms : Meeting notes This page last changed on May 06, 2013 by oreilly. ## 5/25/2013 Met with Alex Worden to discuss her cytometry applications, approaches Attendees: Alex, Jim, Denis, Tom ### ### Tom's notes Jarred is willing to give a talk when he visits Alex' lab in May. Will keep us apprised of the schedule Jarred has grant to develop staining capability for Seaflow/(Sealabel?) - measure grazing by diatoms, dinoflagellates, reproductive rates Very important to engage UW collaborators at high intellectual level (as opposed to "contract labor"). UW has very extensive expertise in oligotrophic cytometry - informatics and biological. Too expensive/daunting to attempt to replicate that... Big science problems: Oligotrophic ocean: what picoplankton organisms are out there? How are the populations changing on various spatial and time-scales? Why are they changing? Only a few cytometers able to resolve picoplankton Need 488 nm excitation for phyto pigments Diurnal growth cycles can now be extracted from Seaflow data, based on new modeling approach. AUV advantages realtime survey capability to inform ship sampling plan 3D measurement capability Long-duration measurements during extended missions Vehicle sensor payload provides contextual data (e.g. nutrient measurements important!) AW would use Cyto-AUV to write papers that utilizes the above advantages Instrument features: Need 488 nm excitation Must resolve pico- and nanoplankton (0.2 - 20 micron) imaging probably not useful - can't discriminate between very small (< 3 micron) organisms, e.g. grazers staining is useful (e.g. for fungi, bacteria, DNA measurement) but not necessary in-situ calibration w/ beads is critical Measurement approaches: Need 10% sigma or better on counting. Note that large counting errors can be amplified when deriving other quantities (e.g. growth rates). Note that medical applications usually require 1% sigma or better (10,000 count) Always add calibration beads to sample to verify alignment, detection, etc (except for DNA samples) Usually able to use single configuration for given seawater sample (detectors have 4-decade dynamic range). May need to change configuration for different water mass (e.g. open-ocean vs eddy?) Not sure how large cells affect Seaflow measurements BD Influx measurements are typically 10 minutes (25 microliters/minute) AW lab does not "gate" on fluorescence or scatter - keep all raw data (may not be practical for coastal waters) Current focus on cells smaller than 5 micron in open ocean, but including larger cells may be important in some environments Prochlorococcus and synechococcus are readily identifiable - eukaryotes more problematic Major challenges: Time consuming, expensive to collect sparse 3D datasets from CTD casts. Seaflow is vast improvement, but surface water only. Data analysis (see below) Data analysis challenges: Cytometer signatures depend on many factors; diverse organism populations, nutrients, depth, etc... Lots of human judgement, calibration, tweaking required to analyze data - difficult to automate (look at Ribalet et al PNAS paper on bio-diversity index) UW team has enormous experience and knowledge - biological combined with informatic Potential alternatives to cytometry - Raman is talked about, no one appears to have used it yet. ### Denis' notes Application: - What groups of organisms are over there? Cytometer on AUV could provide a yo-yo capability, 3D and so on, this means presence. - Day-night dynamics. - Characterize food web is good information is microbiology Observational challenges: - Total throughput from sampling to real data, is very low (presently), sometimes takes years if sample are analyzed in the lab. This also puts a limit on a numbers of samples. - Presence is very low (presently), limited number of ship cruises Target organisms and mixed populations - Not really concerned with high dynamic range on sizes (<5um?). - Discriminate between groups of organisms, using optical methods, Ch A and accessory pigments. Features: - Imaging. Value of imaging is limited, organisms below 5um appear as "blobs", can not distinguish. Low Value. - Real time processing. Normally cytometer is used during cruises by Worden group. On ship, adaptive sampling strategy is used, and decisions on cruise plan are made based on CTD and cytometer data, if it possible at all, and a team have enough bandwidth to do so. High Value. - Bacterial communities need staining. - Population identification. Gating is not used, instead, trigger on scattering signal is used, and then accumulate data with other parameters. This is "see everything" approach. This works well in "blue water". Coastal zones with much more silt etc., is a different story. Value of integration with mobile platform - adaptive behavior, contextual data; real-time data, reconnaissance, surviving; persistent presence, high-res. All High Value. Procedure: - Typically, sample is split in 2, with calibration bids, and without, and both run thru cytometer. - Beads are needed to calibrate optics for sensitivity and alignment. - Typical sampling rage is 25uL/mil, so it takes 10 min to analyze 250 uL; ~ 1000 eukaryotes, number of cyano bacteria's ~ 100x higher. - Statistical errors: <10% is good. - As a "rule of thumb", about 10k cells needs to be accumulated in flow cytometry, in order to compute growth rate, which is difficult. - Gain control: manual, interactive. Use to need gain control adjustment before running some samples, or to resolve different species in the same sample. Now, with 4 orders of dynamic range, only occasional adjustment for regions of ocean are being made. Data analysis. - Not a static process. Interactive process requiring involvement of skillful individual such as scientist. - Signatures of groups of organisms are not static, that is why very helpful to use calibration beads. - There is a lot of knowledge went into data processing in established groups, would be very hard to replicate that. - Interdisciplinary field between biology and computation => high value. Trends: - Imaging cytometry - Smaller particles - Certain interest to Raman, but nobody did it yet. ## 3/27/2013 Sorry these notes are delayed. Denis have I missed anything important? Attendees: Denis, Tom Note that Dorota's "Taxa" table is now online. Reviewed instrument comparison tables on project Confluence page. Consider LISST Holo to be an imaging cytometer; should be included with other existing cytometers Condense some table rows into a single row, with more descriptive text in each cell Good start on science applications/requirements page DK: We are a month behind schedule specified in our proposal - hoped to have "literature search" complete by March 1. DK: As we read articles and attach them, it is useful to markup the PDF file with notes Alex is available next week - review nature of her lab's work beforehand Action items: Denis to review new technologies described in "perspectives" section of Cytotechnology article. Tom to continue instrument comparison tables, filling out science applications/requirements page All: prepare to interview Worden by reading her lab's publications and posters (e.g. this one) ## 3/12/2013 Interview with John Ryan. Ryan, Klimov, Bellingham, O'Reilly Ryan is interested in particle measurements, going back to MUSE-2000 experiment looking at coupling of phytoplankton with re-suspended sediment. Has never used cytometry. Lots of work with LISST-100x and LISST-Holo. LISST-100x yields particle size distributions (volume concentration, NOT particle counts) and shape info. Groups sizes into 32 bins between 1-250 microns. LISST-Holo has been integrated with Dorado. Images particles in 25-2500 micron range. Images are identifiable at least to genus level. Acquires 1 image per 5 seconds. Image generation (30 sec per image?) is currently very time-consuming, performed post-mission. JGB notes that LISST Holo image processing could be much more efficient - e.g. see work by George Barbastathis of MIT In situ Holo processing could enable HAB-seeking AUV LIST 100x processing is also slow, non-realtime. No one has worked to make processing realtime. Ryan, Maughan, Cline are working on processing algorithms (post-mission non-realtime for now) through CANON, contingency time Must make several assumptions for LISST-100x processing; particle transparency, shape... there are also unexplained "blank" anomalies; must use "at sea" blank rather than "in lab" blank - why? (e.g. include dive to off-shelf deep clear water for "blank" sample) LISST-Holo only costs $25K JGB: LISST-100x is very difficult to work with, calibrate due to above problems. Cytometer approach is more direct, requires fewer assumptions. JR: Imaging Flow Cytobot is very cool, not yet integrated with AUV though. JGB: Check out FlowCam - some early problems (e.g. with biofouling) but making progress. Might be able to borrow one for CANON Fall 2013 ## 3/7/2013 - telecon with Holomic LLC Pre-call: Denis - the cell phone cytometer is not prssure-tolerant. Holomic achieves high optical efficiency; can we achieve that? Ketaki Sood (marketing) , Nevan Karlovac (CEO), Ozcan (scientist) O'Reilly, Bellingham, Klimov JGB: Gave MBARI background. Autonomy. Increasing emphasis on microbiology. Physical sampling. Mentioned summer intern program. Ozcan: what about broad-field microscopy? JGB: tricky in situ. Contextual sensors for water samples JGB: What are max flow rates that prototype can sustain? Holomic: Low so far, Microliters per minute... Holomic: Current software gives particle counts, not size Also wide-field fluorescent microscope DK: Unattended operation is key. Low power, size Holomic hasn't focused on low power Holomic: would require significant funding to build instrument for MBARI JGB: MBARI engineering is capable of significant contribution Holomic: formulate your requirments, parameters, etc. Holomic sells products. Cell phone cytometer is not yet a product. JGB: Oceanographic market is quite small. How important is continuous sampling? Holomic: Will keep MBARI in mind as they develop the cytometers Post call: Need to develop requirements - particle densities (concentration) - organism - flow rates, integration time - sensitivity, chlorophyll concentration - excitation/fluorescent wavelengths Compare to BD, Seaflow Need to talk with Alex, Scholin, about the above Example: want to resolve organisms that generate signal detected by EcoPuck; want equivalent sensitivity Send them a thank-you email, invite them to visit For next Tuesday: Target organisms and sizes How to detect (fluorescence, polarization..) Characteristics Natural concentrations JGB: John Ryan is a good resource for the above Meet again in 1.5 weeks - March 19 Ask Dorota what organisms BOGS tracks; she's compiling a table Ask Ryan and Chavez; what organisms would you look for with flow cytometer? Denis and Tom to interview ## 2/26/2013 Action items for next meeting Contact Zhu about cell phone cytometer; latest developments, availability of device - Tom Look at Zhu et al paper to determine feasibility of reproducing device - Denis Consider potential embedded imaging intern project - Tom Review capabilities of Sosik's Imaging Flow Cytobot - Jim Note: Post-doc candidate Eric Rehm will visit MBARI on March 18-19 Let's explore possible Becton-Dickinson tour when Alex returns (she has several contacts there). JGB: Cell phones for robotics are very interesting and promising - high capability, low-cost sensors, computing power Post-doc Eric Rehm will visit on March 18-19 DK: Note that theoretical imaging limit is about 1 micron DK: Variable bandpass filter via electronic control/piezo-electric control JGB: Could pulse multiple frequencies on a given particle to measure multiple parameters Could flow channel be designed to segregate/orient particles based on pressure gradient? Flow "panel" rather than "stream"? Investigate flow cytobot - what have they achieved? Look at citations of Zhu et al: Microfluidic diagnostics for the developing world: http://pubs.rsc.org/en/Content/ArticleLanding/2012/LC/C2LC90022J Millipore Scepter: $3.5K - gives similar data as LISST, which is much more expensive Approaches: Coulter Imaging Flow cytometer Ask Zhu et al if device is available Think about intern project Denis will look into reproducing cell phone camera Review flow cytobot work Becton-Dickinson tour/contact? ## 2/5/2013 Action items for next meeting: 1. Look at cytometers-on-a-chip literature - Jim, Denis, Tom 2. Fix Confluence access - Tom Done - please login into Confluence and verify that you can access https://oceana.mbari.org/confluence/display/CytometerTech/Home 3. Update Confluence "Technologies" page with Polychromator reference - Denis Agenda: 1. Discuss action item status from last meeting (see below) 2. Jim has some information from ONR meeting Denis: American Society for Cell Biology Trade show Cytometery development workshop - every year at Asilomar. Shapiro has been co-chair Denis: Polychromator for flow cytometry (Asahi Spectra) - simplified alignment (about 4x4") ESE Fluo Sens SD - similar assembly for fluorometer Jim: attended ONR meeting; saw presentation on Electro-osmosis Accelerate fluid through a charged tube; potentially eliminate cytometer pump Could you build cytometer-on-a-chip? Could run at ambient pressure Try googling cytometer on a chip; multiple hits. Why can't we use one of these? Denis: Miniature silicone rubber diaphragm pumps on MEMS chip is a common technique Jim: Why are cytometers still pretty complicated and large? Take another look at cell phone cytometer... Check out "google scholar", patent database for "cytometer on a chip" Several home medical devices in development Jim: We should cold-call some of these researchers, ask them questions What are target organisms? Denis: what are minimum throughput requirements, size ranges? Jim: talk with Chavez, Scholin, Worden DAC workshop this week, with Scholin, Delong - we will attend, get an idea of the organism targets Jim: Eric Reihm has been offered post-doc at MBARI, has not accepted yet. Action items from last meeting: 1. Set up project library - Tom Done - confluence page at https://oceana.mbari.org/confluence/display/CytometerTech/Home 2. Refine technology evaluation matrix - Denis Denis will work on this 3. Check for local relevant trade shows - Denis American Society for Cell Biology Trade show. Photonics West - this week Cytometery development workshop - every year at Asilomar. Shapiro has been co-chair 4. Ask Johnson for Coulter counter briefing - Tom Done Done - Ken notes that project has been inactive for serveral months, hopes to restart soon (with Ginger Elrod as chief engineer). See https://oceana.mbari.org/confluence/display/CytometerTech/Coulter+Counter ### 1/18/2013 - Kickoff meeting Action items: 1. Set up project library - Tom 2. Refine technology evaluation matrix - Denis 3. Check for local relevant trade shows - Denis 4. Ask Johnson for Coulter counter briefing - Tom Attendees: Jim, Denis, Tom JGB is recuiting potential post-doc Eric Rehm from University of Washington; he's primarily interested in ocean optics, with extensive software expertise. (Note Rehm's 2007 cytometry publication with Swalwell and Armbrust.) Would be good to arrange Rehm visit to MBARI, before post-doc decision in mid-February. Perhaps he could participate in Jim's DAC workshop to be held Feb 6-7. Jim will pursue travel funding options with George Matsumoto, other sources How to engage MBARI scientists with cytometry? CANON PIs: Worden, Scholin, Chavez. JGB notes that we shouldn't expect a lot of engagement from scientists at least during early phases of feasibility project. Note that Jim is holding a CANON DAC workshop, Feb 6-7; he will see if we can give brief presentation describing our project. Johnson lab is developing a Coulter counter. We should talk with them about details of their project. Note that Ken Johnson is thinking about how AUV skin surface might be used as Coulter counter detector. JGB notes that at minimum Johnson would be great advisor to our project, although he's not too engaged with CANON Tom and Denis discussed meeting/lab-tour from Johnson group - Tom will ask Ken Literature search of existing technologies; we should rank these in evaluation table (refined/codensed version of criteria we presented in proposal) CytoBuoy CytoBot SeaFlow Cell phone Other technologies Denis suggests we check biological, optical trade shows. We should meet about every two weeks.

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