You don't have to have the laser off to measure FRET. This misconception arises probably from microscopy, where you don't have temporally distinguished excitation.... Anyway, the FL3 signal will have your FRET signal irrespective of whether or not the diode laser is on or off--so just leave the laser on. FRET signal will not appear in the FL4 channel anyway--it comes from first laser excitation, therefore you wouldn't see it in the FL4 channel! mr At 1:55 PM -0400 7/1/02, Richard Konz wrote: >Greetings everyone: > >An investigator in my research group is doing FRET by facs analysis >on a two laser FACScalibur [FITC/PE and CY5]. During experimental setup >we use the red diode laser to look at the CY5 staining signal. >However when we do the >FRET experiment we need to have the diode laser off. When you shut >the diode laser off, >Cell Quest will eliminate FL4 as a collection parameter. > >Thus far, we are unplugging the diode laser from the board while we >are doing the FRET >experiment. I could install a switch to cut the power, but in a >multi-user facility >this would probably cause problems with the users forgetting to turn >it back on. > >Does anyone have any ideas or potential workarounds with this FRET >combination? > >Thanks for your input and all my best regards, > >-Rich > > > >Richard F. Konz, Jr. >Scientist >Manager, Flow and Image Cytometry Facility >Wyeth >200 Cambridge Park Drive >Cambridge, MA 02140 USA >Office: 617-665-5522 >Lab: 617-665-5551
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