Geoff, To start from the end. > A friend of mine with a new Vantage SE uses a 1.5 drop envelope I > dont see the logic in this as half the time it sorts 2 drops and half > the time it sorts 1!Which is best? When Vantage sorts on 1.5 drops it does not randomly sort 1 or 2 drops. Logic is that it virtually divides drop (i.e. drop time) in 10 equal sections. If it's estimated that target cell is going to end up in first 3 sections, that drop and previous one is sorted. Accordingly, if it's in the last 3 sectors following drop goes instead and if it's in central 4 sections only 1 drop is sorted. I use 1.5 drops in most of the cases, gives me good purities and keeps aborts less than 10% on 1 cell/6 drops, meaning 5000 cells/sec. on 30KHz, 11PSI which is quite fast for "low speed" with acceptable recovery. Of course recovery is not an issue for CloneCyt and you should go for 3 drops always. > I'm sure lots of people reading this with > ageing Vantages get some varation in drop delay it seems to me this > is being compensated for by a three drop envlope I had honour to have a lesson from Mr "Vantage Designer" Mike Hoffman and if I understood him right, difference between what you calculate and real drop delay comes from the fact that dead time (>5µs, adjustable) and sort decision time (0.5µs pulse) are not included in simple calculation that takes break-off distance (delay time) as a whole, as if machine starts "delaying" immediately from interception point. There is no reason that by ageing Vantage would change this difference, but any major fluidics repairs and/or electronics boards replacement could affect this as well as playing with dead time or using pulse processing. My system has -0.3 offset on 30KHz which could be explained by ~10µs excluded from 1/30000 sec. drop = 33µs. Old Vantages have bigger offsets because they have longer dead time and much longer processing time if they were upgraded with SEM. Just for clarification, Sasha. •••••••••••• Dr Sasha Sreckovic Dept Path & Micro University of Bristol University Walk Bristol, BS8 1TD, UK Sasa.Sreckovic@bristol.ac.uk +44-(0)117-928-8606 •••••••••••• ---------- >From: "geoff.morgan" <geoff.morgan@bbsrc.ac.uk> >To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >Subject: Clone Cyt-How many drops >Date: Thu, Feb 10, 2000, 1:09 pm > > > Dear flowers, Mike Evans's logic seems right if the machine is set up right > a single drop envelope with one cell in it should work. > Two points 1.In the Clone Cyt user guide it only says optimise the > sorter for sorting. On page 24 it shows the sort control window with 3 > deflected drops and counter Sort Mode. 2.In the Vantage user guide when > in Counter mode the 3 drop envelope(shown in the picture) is extended to > 4 "to achive high count accuracy" From my own experience a three drop > envelope gives better purity over a wider range of Dop Delays than a > 2 or 1 drop delay.With a one drop envelope drop delay must be spot on, > any drift and purity is lost.I'm sure lots of people reading this with > ageing Vantages get some varation in drop delay it seems to me this > is being compensated for by a three drop envlope.What do other people > use? A friend of mine with a new Vantage SE uses a 1.5 drop envelope I > dont see the logic in this as half the time it sorts 2 drops and half > the time it sorts 1!Which is best? > > > Geoff Morgan Babraham, Cambridge UK > > m
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