Dear Colleagues, Thanks for all the advice for my efforts to get a decent Cy5 signal in Fl4 on my FACSTAR Plus. Here's what worked: Initially I asked the question because I couldn't understand why I could not duplicate the second laser conditions on the Calibur. I can pump the dye head with up to to 5W and get a full 1W at 600nm from the 599 dye head and therefore assumed - based on the rhodamine 6G power frequency curve - that I could use the birefringent filter to set the dye head into the red with only a nominal loss of power. I did this and didn't really get much effect on the Cy5. At 100mw maybe a quarter log so my question was really aimed at some mystery intrinsic difference that I was missing between a 635nm diode and a ~635nm dye head. Thanks to all of you who suggested I take a closer look at the spectral curve the real improvement in signal occured when I replaced the 660/30 bp with a 675/20, about a half log, and in fact David Adams at Systemix corroborated this, this morning. Gene /UCONN Health
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