Hi Andrew, I too have seen a lower signal on a FACSVantage SE TSO in the FL3 detector when compared to other BD instruments. If you have Optics configuration 6, the Vantage SE is fitted with JUST the FL3 detector, not the FL3/Flx (Optics configuration 7, detector option 5 laser 1) configuration, the PMT housing is bolted directly to the main housing with no relay lens prior to the FL3 detector, and the light striking the FL3 is therefore "sub-optimally focussed". This results in a slight loss of performance with this configuration. I think I actually talked with Gary Rundle at BD about this more that a year ago and I thought at that time he agree this was so... That said, I wouldn't expect it to be as bad as you describe and I recommend really checking your alignment closely, if you can't completely close the iris and see almost minimal loss of signal in FL1/FL2 then that's an indication that things may be off. Also, try moving the stream position back slightly such that the stream is no longer in the front 1/3rd of the waste aspirator when the instrument is aligned. Then by adjusting the fluorescence height and focus, you can sometimes get a bit more performance out of the instrument. Hope some of this helps, and that it's clear. Geoff At 02:04 PM 12/19/00 -0500, you wrote: > >This message is directed towards FACSVantage users that have upgraded their >machine from a FACSVantage to a FACSVantageSE w/Turbosort. I am >experiencing a >decreased sensitivity in FL-3. When comparing alignment particles (cRBC, >Alignflow beads, Calibrite beads) between a FACSVantage, FACSVantageSE w/TSO >and >a FACSCalibur, results have shown the FACSVantage SE signal to be >significantly >lower than the Vantage or Calibur. Furthermore, when looking at splenocytes >stained with CD4 PE-Cy7 (787/40 filter), there is no discrimination between >negative/unstained cells vs. stained cells. The signals on the FACSVantage >and >Calibur are nearing the third decade while the FACSVantageSE signal was >negative >with log amps and voltages greater than 900V. I tried increasing the log >offset >as far as it will go and found little if any improvement in the signal. > >Boards have been replaced and new PMTs in FL3 have done nothing to remedy >the >problem. BD has been very responsive to my concerns, but the problem is >still >present and I was wondering if anyone else has experienced similar problems >with >the extra mirror and different optical path for a 5-color FACSVantage SE. >Any >comments or suggestions would be greatly appreciated. > > >Andy Morschauser >Schering-Plough Research Institute >Department of Immunology >2015 Galloping Hill Road >K15-3-3945 >Kenilworth, NJ 07033 >908-740-3089 > > Geoffrey Osborne Specialist, Flow Cytometry, John Curtin School of Medical Research, The Australian National University, Canberra, 0200, ACT. AUSTRALIA email: geoff.osborne@anu.edu.au http://jcsmr.anu.edu.au/facslab/facshome.html
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