Mark Olsen writes: > >I am a chemistry grad student, and my project is to sort genetically >engineered libraries of E. coli. I am currently investigating ways to >increase the rate of sorting on a commercial droplet sorter. Most of the >commercial machines have a 76um orifice, which is much bigger than coli or >yeast. I have noticed a reference from Shapiro's Practical Flow Cytometry >(3rd) by Fellner-Feldegg on using a small 25um orifice. Is it realistic to >fit a BD Vantage or a Coulter Altra with say a 20um orifice and a 100kHz >droplet generation frequency, and thereby increase the effective sort rate? >I am guessing that a cycle time of 5.5 microseconds means a maximum of >180,000 decisions/sec. This seems acceptable? I noticed that the >Cytomation MoFlow uses droplet deflection technology, but would the BD or >Coulter drop deflection plates result in acceptable purity and yield? Is >jet-in-air required for these kinds of sort rates? Are there other >considerations that need to be taken into account? I apologize for the >ignorance, and thanks in advance. > The orifice size is not of particular relevance here; it is the droplet generation frequency which will determine the maximum sort rate, up to the point at which the instrument electronics can't keep up with the required decision rate. If the processing dead time or cycle time is 5.5 microseconds, the 180,000 decisions/second is what would be achieved if you had a steady stream of cells coming one right after the other; in practice, you need to have a lower duty cycle, which among other things, will keep the coincidence abort rate at an acceptably low level. Thus, the practical processing rate of an instrument is generally lower than would be calculated from its cycle time. But worrying about orifice sizes and droplet generation rates first is going about things ass backwards. You've said that you want to sort genetically engineered libraries of E. coli. What marker(s) will you use to identify the cells you want to sort? What is the frequency of desired cells in the population? What are you going to do next with the sorted cells? Do you need single organisms (neglecting, for the moment, the possibility that two or more bugs might be stuck together and look to the instrument like a single organism)? Does your lab, department, or institution already have the sorter or are you looking to buy one? From your question, it appears that you are considering modifying a B-D or Coulter instrument to run beyond specifications, to achieve performance closer to that specified for Cytomation's system. But, as I said, you need to start with the biological details of what you're doing. Would your project be feasible if you could sort 100,000 cells/second and not if you could only sort 20,000 cells/second, which only takes five times as long (and let's face it, while time may be money, graduate students' time is usually considered to be in the category of small change)? Defining all aspects of the problem precisely will, at least in my opinion, be essential if you are trying to get funding to acquire a high-speed sorter to do this project; there are people who know nothing about flow who can snap their fingers and have the bucks for a high-speed sorter appear, but most of them have also dined with the King of Sweden in December. I hope the further discussion of this subject on the Mailing List doesn't degenerate into "My nozzle has higher pressure than yours", but I can be contacted off the List if necessary. So, let us know more about your problem. -Howard
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