Gene Pizzo wrote: Furthermore, why on the FACSCalibur is it necessary to align the two beams in time when on the FACStar Plus the electronic separation of the two beams in time is considered a necessity? The difference between the Calibur and the FACStar Plus is that on the Calibur (with a 2nd laser) you are trying to detect two dyes that, if interlaser compensation is not done, will both generate a signal in the same (FL4) PMT. Specifically, you are trying to use PE-Cy5 and APC simultaneously; the Cy5 component of the PE-Cy5 dye will be excited by the 633nm laser and generate a signal in FL4. What you are doing is actually adjusting the time that the signal from the 1st laser (PE-Cy5) is delayed in order to subtract it (compensate) from the signal off of the 2nd laser (Cy5 component of PE-Cy5). You are not, as I think you are suggesting, aligning the two beams to be colinear; they are still separated in time. Kevin L. Holmes, Ph.D. Head, Flow cytometry Unit Office of the Scientific Director Bldg. 7, Room 01 NIAID, NIH Phone: 301-496-9071 FAX: 301-402-4532 Email: kholmes@atlas.niaid.nih.gov Colleagues, I'll ask the question again...it hasn't been answered here yet, twice. It wasn't answered in BD class when I asked it. It wasn't answered by the service engineers I've asked. Maybe no one has an answer sort of like that question in computer class years ago "does 'bit' derive from the first two letters of binary or the last two letters of digit?" We all understand that the Cy5 of the PE-Cy5 tandem can be excited by the red laser, that's the easy part. But if the signal generated from the two beams is separated in both space and time why should there be a compensation necessity? Furthermore, why on the FACSCalibur is it necessary to align the two beams in time when on the FACStar Plus the electronic separation of the two beams in time is considered a necessity? Gene Pizzo/UCONN Health
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