Hello Glenn, My guess is that your time delay setting (ie. gap between your 488nm beam and red beam) is close to the right. For a bigger particle it will take longer time to fully go trough the first laser beam ie. shorter time delay between 1st and second laser when compared to a measurement done with smaller particles. In Calibur the dual laser timing can be wrong only (+-)22us. We know that the mean velocity in flow cell is 6m/s so the lasers can be wrong only (+-)132um to make time delay calibration to finish succesfully. If you want to use smaller beads for calibration you should probably lift your red laser beam slightly closer to the argon beam (in theory 6um-2,5um=3,5um the truth might be 10-20um) Thats my guess, hope that you solve the problem! Mika PS. No guarantee given.. Mime-Version: 1.0 Date: Tue, 3 Oct 2000 17:00:40 -0400 To: cyto-inbox From: Glenn Paradis <gap@MIT.EDU> Subject: time delay calibration particle X-PMFLAGS: 34078848 0 1 18254.cnm X-Orcpt: rfc822;mika.korkeamaki@utu.fi Status: Hi Everyone, 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. Glenn MIT Mika Korkeamäki Project Engineer BioCity Turku Office: Dept. of Medical Microbiology e-mail: mika.korkeamaki@utu.fi Kiinamyllynkatu 13 phone: +358- 2-333 7563 mobile: +358-50-564 6855 20520 TURKU fax: +358- 2-233 0008 FINLAND
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