James, Whereas the problems you see could certainly be due to bad luck with the diode lasers, it is also possible that the mount for the red laser is giving you problems. We have noticed that the extra weight of the new TEC diode laser makes it more vulnerable to becoming misaligned, resulting in loss of signal and in loss of ability to compensate because the timing of FL4 pulse is out of the time delay calibration window. To distinguish the new possibilities, you (or your service rep) can measure the output of the laser (if lower than specifications, obviously it needs replacement); if the power is OK, then check time delay calibration. If time delay fails and the PMT voltage required to meet the target channel is higher than normal, the laser alignment may have drifted. Time delay calibration is a sensitive measurement of how the instrument is functioning, and even a small misalignment of the laser will prevent it from passing. Kevin Kevin L. Holmes, Ph.D. Chief, Flow Cytometry Section Research Technologies Branch Bldg. 7, Room 01 NIAID, NIH -----Original Message----- From: James F. George, Ph.D. [mailto:jgeorge@uab.edu] Sent: Monday, December 17, 2001 5:26 PM To: cyto-inbox Subject: FACSCalibur Red Diode Laser Reliability Greetings fellow flow cytometrists We recently acquired a new dual laser facscalibur. We have had some serious problems with the stability of the red diode laser. In two months, the laser has been changed out three times and our fourth service call for the same problem will commence tomorrow. Typically, one will observe a decline in sensitivity in fluorescence in channels that rely on the use of the diode laser, accompanied by a large increase in the amount of compensation required for those same fluorescence channels (I am being deliberately non-specific here to accomodate those who do not have BD instruments, for those facscalibur users, I am obviously talking about FL4, but this would depend on the fluorochromes that were being used etc). I went with BD because my previous long experience with the highly reliable Facsscan and the fact that I really needed two lasers. Unfortunately, the other laser is not useful if it is unreliable. Have others experienced this problem, particularly since BD switched suppliers for the diode laser? This is making me crazy. Any info you have on this would be useful. I am working with BD on this (upper management has been pretty responsive), but currently I have not seen a permanent solution to the problem. -James
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