Joseph, I can't provide an explanation (other than 'sounds like a digital processing artifact--perhaps brought on by signals near clipping height and accumulating rapidly.'), but since I'll be doing a workshop at Montpellier on cytometry basics on the current generation of hardware/software, I'll be grateful for any comments that will help provide answers at that time. ================ David M Coder, Ph.D. Consultant in Cytometry Seattle WA Cell/Msg: 206 499 3446 Email: d_coder@msn.com -----Original Message----- From: Joseph Webster [mailto:J.Webster@centenary.usyd.edu.au] Sent: Monday, March 01, 2004 8:00 PM To: cyto-inbox Subject: FACS DiVa question Dear All, Has anyone seen the negative cells disappear off the bottom of the scale? We have a 3-laser DiVa, 488+600(dye)+UV. We have observed this using DAPI as a "dead cell" label excited by the third (UV) beam, but have also seen it with very bright PE from the primary laser. Users here commonly run with a DAPI (or PI) concentration that puts dead cells at the top of the scale, sometimes over the top. We (usually) see the "live" cells in the bottom decade, a smear in the middle, and a peak of "dead" cells at the top. We recently noticed that the "live" peak is sometimes dropping off the bottom! The middle and high intensity signals don't seem to change, only the negative. More tests to do, maybe the proportions are changing also... The effect is dependent on sample flow rate &/or differential pressure; it has been made to appear and disappear by winding the differential pressure up and down. It only shows up when there is signal at or near the top of the scale. Comparing two tubes in the same experiment, one with moderate intensity PE, the other with very bright PE, we could make the negatives drop off the bottom when there was bright PE present, but in the tube with lower PE it would not happen. It may be affected by alignment or focus, don't know yet. Maybe someone with DiVa experience can tell us what's happening &/or how to stop it. Don't know if other DSP-based systems show anything similar, I have not seen it in any of our analogue instruments... There's always something new to learn in this game! Joseph. -- Joseph Webster, Flow Cytometry Facility, Centenary Institute Locked Bag No.6, Newtown, NSW 2042, AUSTRALIA. Ph: 61-2-9565-6110 Fax: 61-2-9565-6101 e-mail: J.Webster@centenary.usyd.edu.auReceived on Wed Mar 3 11:58:00 2004
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