time delay calibration particle
gap at MIT.EDU
Fri Oct 13 15:58:49 EST 2000
I have found the answer to my question about whether the size of the bead
has anything to do with an improper time delay calibration on our FACS
Calibur. The answer is that yes the size of the particle has an effect.
You should have the FSC gain set to E00 for the time delay calibration or
else the time delay electronics will lock the system out of performing the
calibration. This is because the smaller bead introduces an offset time
component which will cause an improper delay.
I would like to thank both BD and Molecular Probes for helping me to solve
this problem. After calling BD for instrument support and not recieving a
satisfactory answer, I asked the cytometry mailing list for help. A BD
engineer who was monitoring the list emailed me and explained why I was
having trouble with the 2.0 um beads. Also, BD called me twice make sure I
had a satisfactory answer.
Mol. Probes did not hesitate to send me some 6.0um 633 excitation AlignFlow
beads free of charge to test my theory. Of course these beads calibrated
properly and I believe they are the best product I have seen so far for
doing any red laser alignment. The signals are strong and the %CV's are
Lastly, thanks to everyone that made suggestions to my initial email.
>I have a new twist to the problem of an improper time delay calibration on
>our FACS Calibur. I have isolated the problem to the beads I am using to
>perform the time delay calibration and am wondering if someone has an
>explanation for my observation.
>I have been using an old lot of 6.0 um Polyscience beads which is now
>running out. The time delay calibration has been and still is working
>flawlessly with the old Polyscience beads. I recently purchased 633nm
>excitation, 2.5 um Molecular Probes AlignFlow beads A-7312. The beads
>excite very well with the diode laser and are much brighter the the
>Polyscience beads. After increasing the FSC gain to put the beads in
>channel 400 and decreasing the Fl4 PMT voltage to put the beads in the
>third log decade I get an error saying the event rate is too low for the
>Fl4 fluorescence. By looking at the histograms, there are clearly lots of
>beads running through the cytometer and the counters say that the number
>of beads is 1,500/sec.
>When I open the FACS Calibur hood I see the yellow light on the time delay
>board is shining brightly. I have tried running in lo, med, and hi with
>no successful calibration. I have tried resetting the time delay switches
>on the board and nothing seems to help. As soon as I put my old beads
>back on with my normal settings, I get a nice happy beep telling me my
>time delay calibration has worked.
>Is there a minimum size particle that must be used in order to time delay
>calibrate? Any ideas would be appreciated.
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