From: Darren Hickerson (DHICKERSON@brody.med.ecu.edu)
Date: Wed Feb 04 1998 - 17:26:02 EST
Kirk & others: I am using calculated ratios on a FACScan using a visible laser Ca technique analogous to Indo-1. I have compared the results to those obtained on FCS Assistant. The biggest problem I see with FCS Assistant is that it seems to use the superimposed "1024" channel axis that the data is (are) actually saved in to calculate the ratios, rather than the 10,000 channel true fluorescence scale that reflcets actual intensity values, so the final ratios are not "real" values. The pictures are very nice, but the numbers don't reflect the real changes. Hand-calculating the intensities seems to be the best way (true FL"A" divided by true FL"B" for a given time point -- you just lose the flow of the kinetic scale this way). Phoenix Flow has some software that SEEMS like it would do this for you (from the pix on the ads), but I'm not sure. Anyone else with advise or how to get "real" ratios on FCS Assistant, or with more detailed info on Phoenix software, please write in. Also, if anyone has a quick "how-to" on setting up the real ratio parameters on a FACStar Plus, I'm all ears. The manual gets so bogged down in calibrating the axis, I'm not sure if just setting it so it "looks good" even works. Same questions again: can I use "log" scale FL parameters, or do they all have to be set to the 1024 scale before acquisition? I asked this before & never got clear on what people were calling linear, log, channel, and "true fluorescence" values -- everyone seems to use different terminology. Thanks, Darren Hickerson dhickerson@brody.med.ecu.edu Core Flow Cytometry Facility, Brody 4W37 Department of Microbiology and Immunology East Carolina University School of Medicine Greenville, NC 27858 Phone: (919) 816-2799 Fax: (919) 816-5018
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