901303 Cytometer Technologies for Autonomous Platforms : CytoSense, CytoBuoy, CytoSub This page last changed on Apr 23, 2013 by oreilly. Summary from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3279584/ : CytoBuoy b.v. is a dutch company which manufactures particle analysis instruments and software for mainly aquatic and marine science. Since 2001, the bench top scanning flow cytometer CytoSense is designed for pico- , nano- and micro-plankton studies. It combines classical flow cytometry data with silico-images of the measured particles and targeted video imaging. This instrument is equipped with 2 lasers (460 or 488 or 532 or 561 nm and 445 or 635 or 640 or 660 nm) and up to 10 detectors. The CytoSense works with a hydrodynamic sheath fluid injection system with external and recirculating mode, and an auto-adaptive speed controlled from 0.2 to 20 µL/s. The CytoSense may be extended with a video imaging-in-flow at rates of up to 1,000 scans per second. The CytoSub module "Shallow" allows a submerged use in shallow waters (depth of 20 meter max.) and CytoSub module "Deep" transforms the CytoSense flow cytometer into a CytoSub instrument for submerged operation down to 200 meters. The Buoy module transforms the CytoSense into a CytoBuoy analyzer for moored operation, with 8 solar panels systems with rechargeable batteries and flashlight, telemetry and Argos transponder. CytoUSB software is used for instrument operation and storage of measured data files (8 bits---3.5 decades). CytoClus software is used for data analysis. Thyssen et al(2008) evaluated CytoSub performance in the Bay of Marseille and the southern Indian Ocean; they compared CytoSub data to data acquired by "conventional" cytometers. They note that limitations in instrument memory and sampled volume can lead to undersampling, but describe a workaround to overcome this. (This problem might not apply to present-day CytoSub instruments). The prototype CytoSub instrument was integrated with AutoSub by Alex Cunningham's team at the Environmental Optics Lab in Glasgow, in 2002 (presentation, paper1, paper2). The prototype has since been refined to today's commercial CytoSub, with smaller size, better performance, and imaging capability. But CytoSub hasn't been deployed on another AUV since AutoSub (according to CytoSense CEO George Dubelaar). More technical info from CytoSense Dominic Stuart: Flow Rates Our sample pump has a range of 0.07ul/s up to 18ul/s or 0.1ul/s up to 30ul/s (depending on the tubing used in our sample pump). However the big speeds of 18 - 30ul/sec are usualy only used for flushing purposes or searching for very big organisms. The speed of the sample pump affects 2 things. 1. Particles/second If you increase the sample pump speed, you will push more sample through the machine which means that the amount of particles per second increases. Our current system can process up to 5000 particles/second (small particles and a standard 3 detector system). If the concentration is too high, the sample pump speed needs to be lowered for the system to handle the datastream. 2. The Sample core The sample pump pushes the sample into the CytoSense. When it reaches the injector, the sheath fluid "pulls" The sample fluid through the cuvette where the laser scans each particle one by one. The sample core is aligned by the sheath fluid. In the center of the sample core, the optics are the most sensitive and this is the "sweet-spot" where you would like the particles you are analyzing. If you increase the sample pump speed, the sample core gets wider, this means that the position of the particles in the laser beam are not always optimal and you can notice shifts in your data for small particles (this is less important for big particles because a part of them will always be in the center). 3. Conclusion It really comes down to: a) What are the size ranges you are interested in? (bigger things, are generally less abundant and sample core width is also less important, and can be analyzed at higher flow speeds) What we would suggest, if you would like to have a broad analysis of your sample, is to perform say 3 measurements at different speeds and trigger levels. This results in 3 data files where you get the best of both worlds (accurate data for your small particles and enough big particles). Roughly: Sample speed 0.07 - 4 ul/sec - Picoplankton, bacteria Sample speed 4 - 12 phytoplankton Sample speed >12 big stuff (low abundant) Digitizing and PMT Events The electronics of the CytoSense digitize the data from the PMT's this works as follows: 1. Event We specify an event as one single triggered data scan (across all channels) which is normally 1 particle unless the concentration is too high or the trigger level is too low. One event varies between 100 and 100K bytes depending on the configuration of the machine. Note that you get a datapoint every 0.5um on every channel.Examples for a 10 channel machine: A particle of 2000um = 4000 bytes * 12 = 48000 bytes (46KB). A particle of 10um = 20 bytes * 12 = 240 bytes. 2. Triggering The trigger level and the trigger channel (PMT) can be set in software. The electronics will monitor the selected trigger channel and if the PMT output level is higher then the set trigger, the electronics will start to capture and digitize all channels (PMT's). The electronics capture the data at 4MHz, combined with the speed of the particles shooting through the laser (2m/s) this results in a datapoint for all channels every 0.5um. Once the trigger level drops below the set trigger level, the electronics stop capturing and will consider that event as 1 particle. 3. Limits The electronics can capture and process 5000 particles/second (small particles and a standard 3 detector system). If you have extra PMT's in your configuration, you generate more data with your machine and this number will be a bit less. However, we are currently working on new and quicker electronics! The electronic limit of 5K events/sec is not the only limitation of the system. Because of how our triggering system works, there needs to be a "gap" between particles. The electronics need to detect the end of 1 particles before it can detect the beginning of the next particle. If you pump to many particles/second into the machine, the system will eventually not be able to process all the particles because they are so packed together that the system will interpret it as one big particle. 4. Conclusion The maximum amount of partciles (PMT events) the system can process is 5000 events/second. SUB Power consumption The amount of power varies depending on the configuration of your instrument (how many pmt's,how many lasers, high power laser, imaging in flow etc etc. To give you a general idea, if you have a full option machine during a measurement the instrument will peak at +/- 60W. When the system is powered but idle, the system will draw +/- 20W. Between measurements the system can entered a preprogrammed deep sleep, during this phase the system will draw +/- 2W. I Attachments: CytoSub-AutoSub.pdf (application/x-pdf) CytoSub-AutoSub-2.pdf (application/x-pdf) CytosenseSpecifications.pdf (application/x-pdf) Cunningham-CytoSub-slides.ppt (application/mspowerpoint)

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