I agree with Joe Trotter that the "3 puddle" method is the accurate way to determine the drop delay. However, with the same number of outliers in the ?1 and +1 offsets you could still have a very low recovery. It depends on what that same number is. In Matt’s case I estimate to find 30 outliers in each offset (60 outliers total) when you use the "3 puddle" method at 500 Ev/s and sort 1500 events using a 1.0 drop envelope. Which (I believe) is reasonable for a Vantage with TurboSort. Ofcourse, in theory you shouldn’t find any outliers. Unfortunately, ‘fluidics’ is one of the major factors that affect a perfect drop delay. An event of interest doesn’t always end up on the location that the sorter predicts after it has been detected. Apparently, a particle sometimes travels a littlebit faster or slower on its way to the ‘last attached’ drop. The less stable the fluidics the more you expect this to happen thus more outliers show up in the two offsets. One way to minimize the risk of having an event end up in the wrong drop is to increase the amplitude of the dropdrive and shorten the distance between the detection point and the drop breakoff point. It is easy to find the optimum distance by adjusting the position of the laser (detection point closer to the orifice) and increasing the dropdrive to the point just before detecting light scatter caused by vibrations in the stream from the droplet formation. When optimized, it should be possible to find as low as 14 outliers in only one of the two offsets (the other offset should be empty) using the "3 puddle" method at 500 ev/s sorting 1500 events total (> 99% recovery). Ruud -- * * * * * * * * * * * * * * * * * * * * * * * * * Life is either a daring adventure or nothing. (Helen Keller) * * * * * * * * * * * * * * * * * * * * * * * * * R. Hulspas, Ph.D. Head, Cancer Center Flow Cytometry Lab UMass Cancer Center UMass Worcester Foundation Campus 222 Maple Ave Rose/Reed Building, Rm 104 Shrewsbury, MA 01545 phone: 508 856 1604 fax: 508 856 1310 http://www.umassmed.edu/cancercenter/research/cytometry.cfm
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