Dear All, Well, I discovered I am not alone. I received many responses to my query regarding problems with time delay calibration and FACS Caliburs. Many of the detailed responses were copied to the list as well. Before I summarize I'd like to add a disclaimer that what follows is as I understand the mechanics of time delay, and I welcome corrections from those with greater technical expertise. 1. As I understand it, time delay calibration synchronizes the the pulses coming from first the red and then the blue laser. The system is designed such that the machine expects the time between the pulses to be about 22 microseconds. If either the time varies greatly from that value or is not steady, one gets the "signal out of range" error message. 2. Time delay is important in order to have stable compensation settings. As compensation is done in an analog fashion, an appropriate negative pulse must be generated at the correct time. If time delay is not calibrated, one can see increased compensation values. Failure to set time delay will not cause data from one event to be attached to another. If you are acquiring events at 3000/second, events are still 333 microseconds apart. (compare to 22 microseconds between lasers). 3. If one cannot set time delay, one can either try to compensate (expect higher values) or acquire without compensation and use software compensation using FlowJo. 4. The time it takes for a particle (or cell) to travel between the beams is of course very sensitive to stable sheath fluid pressure. Most of the responses I got were focused on this issue. Specific suggestions were: a) Make sure the sheath fluid cap is on tight and the tubes not twisted. Get the latest BD tank which apparently is made of thicker plastic. b) Make sure the tank is not too full or empty. c) Try replacing the sheath filter. BD confirms that this has fixed the problem on several machines. d) Consider getting a pressure gauge. The machine should be about 4.5 psi. e) Run 10% bleach bypassing the filter as you would for routine maintenance. f) Make sure there is not air in the system. Reprime, tap the filter, etc. g) Make sure your tank is not cracked and your "O" rings are intact. h) Check the bolts holding down the plate over the sheath tank. 5. Fewer responses focused on realigning the laser. 6. Try fresh beads. 7. Set time delay each time the system is depressurized (as when changing the sheath fluid). If a small air leak developed unnoticed, your samples may no longer be appropriately compensated (thanks to Simon Monard for this excellent advice). 8. Using two buttons under the hood, one can "reset" the machine, instructing it to take the current timing between the pulses as the new standard (say 25 microseconds instead of 22 microseconds). As pictures would be required, I suggest that users ask their BD rep to take them through this. You must be acquiring events using the red laser at the time the reset procedure is done. I hope this was helpful. Thanks again to all who responded. Warren Warren D. Shlomchik, M.D. Assistant Professor of Medicine Yale University School of Medicine Section of Medical Oncology 333 Cedar Street WWW217 New Haven, CT 06520 phone:(203)737-2478 FAX: (520)395-8571
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