I've seen this before. Tricolor/cychrome/PE-CY5, whatever you want to call it is a tandem conjugate. The idea is that the 488 laser excites the PE, it fluoresces orange-red and this is in turn absorbed by the CY5 portion of the tandem molecule and then it flouresces further red. This energy transfer is not 100% efficient so you can get significant PE fluorescence showing up in FL2. I'm gathering from your question that you don't have a problem compensating FL2-FL3. Compensation is always more difficult when you have two stains that are vastly different in relative intensity. The more disparity, the more compensation needed. Your FL4-%FL3 problem is exacerbated by the fact that it isn't JUST a compensation problem. Compensation is used to remove the fluorescence contribution due to spectral overlap among fluors. For instance, you have to compensate your "green"(FITC/FL1) dye from the "orange" (PE/FL2)channel because it has an orange "tail". We express this as FL2-30%FL1, for example. What we're saying is that an amount of light equal to 30% of the light detected in FL1 is showing up in FL2 and we need to ignore that much of the contribution to signal in that detector. The problem you're seeing isn't due to to spectral overlap. You see, the CY5 portion of your tandem dye, tricolor, is excited very well by your little red diode laser. It's "real" fluorescence in FL4. (Imagine trying to compensate PE fluorescence out of FL2. It doesn't work so well.) So, the reason your FL4(APC?) signal is being ablated is because it is lower than the siganl you are getting from tricolor. If you compensate the tricolor signal in FL4, you are knocking your dim FL4 signal off scale. You are also correct that there is a time delay calculation that has to be performed by the cytometer to ensure that signal collected when cells strike the first laser is correlated with signal from excitation by the second. You should run FACSComp and do the time delay calibration every time you are using the diode laser. If you're not using FL4, turn it off. And that's all I have to say about that. David McFarland SmithKline Beecham Pharmaceuticals ---------------------- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 07/27/2000 18:14 --------------------------- David_Topham@urmc.rochester.edu on 25-Jul-2000 11:42 To: cyto-inbox cc: (bcc: David C McFarland/DEV/PHRD/SB_PLC) Subject: FL3/FL4 compensation on a Calibur Dear flow cytometrists: We have recently become aware of an interesting artifact regarding 4-color samples on our Calibur. The problem occurs when analyzing samples stained with a bright FL3 fluorochrome, say CD8-tricolor, and a weak FL4 stain, say CD25-biotin/SA-APC. When compensation is set properly with the single color reagents, the weakly staining CD25+/CD8+ population appears to be CD25 negative. The brighter the stain in FL3, the more negative the FL4+ cells appear. So CD25 appears OK on the CD8-negative population. We know this is not correct because when another color is used for the CD25, say CD25-PE, it appears just fine. I believe there is a time delay for the FL4 detector, and I wondered whether this factors into the compensation issue. I have also noticed that it is nearly impossible to overcompensate FL3-FL4, i.e. make the events squish against the axis. We are still experimenting with the issue to try to resolve it with different settings, but I was hoping someone could explain it to me a little more clearly. David J. Topham, Ph.D. Center for Vaccine Biology & Immunology Aab Institute for Biomedical Sciences University of Rochester Medical Center 601 Elmwood Avenue, Box 609 Rochester, NY 14642-8609 Tel. 716 273-1403 FAX 716 756-5614 E-mail: David_Topham@urmc.rochester.edu
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