time delay and FACS Calibur summary
Warren D. Shlomchik
wds8 at email.med.yale.edu
Mon Jul 24 07:34:32 EST 2000
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
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 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
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