Jim, I suppose that if you co-layer the UV and HeNe you will have a working solution but there are other considerations. I and others do not want to sacrifice the HeNe APC signal along with Hoechst. Your solution would not allow this as Red fluorescence of Hoechst would interfere with Red fluorescence of APC. I think that another pinhole in front of PMT to allow for UV signal would be sufficient. This is the kind of problem we need to address with B.D. to improve on the standard configuration. I would also like to use LSR for four dyes off of Argon and two off of HeNe for 6-color. David If you co-layer the UV and the HeNe in the 3rd position you can detect the red signal from the HO dye in FL3. My LSR is configured with a change in laser alignment and pinholes to allow UV/HeNe laser to be seen in FL3. Not so hard to do. Contact BD if unable to re-align yourself. Jim Houston > ---------- > From: David Dombkowski > Sent: Monday, March 4, 2002 7:32 AM > To: Cytometry Mailing List > Subject: SP with LSR > > > Kimmo, > > You will not see red fluorescence with Hoechst 33342 with the > configuration of 2 PMT UV, 3 or 4 PMT Argon and 1 PMT He-Ne. The red > fluorescence is too high for dichroic to reflect to PMT for UV. You need > to > be able to collect this signal in PMT usually dedicated to Argon or He-Ne > laser. There is no pinhole for UV fluorescence in these channels. You need > a custon configuration which may be achieved if you cantact Becton > Dickinson. > > David David M. Dombkowski dombkowski@helix.mgh.harvard.edu Flow Cytometry-Pathology-CNY rm7017 149 13th Street Massachusetts General Hospital-East Charlestown, MA 02129 Tel. (617)726-1683 Fax.(617)724-3164
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