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I just mounted my FL4 PMT 1/4 inch (approx.) higher and got rid
of all the scattered laser light from the 1st laser and it
increased the 2nd laser fluorescence by more than 100-fold!!!
So my signal-to-noise increased by about 1000-fold! When I
took the beam expander out of the laser focusing optic,
fluorescence increased 8-fold. My CVs are between 4 and 6% but
I spent very little time trying to optimize anything other
than FL intensity.
The 2nd laser was placed at 20 microsecs after the 1st in all cases.
So it's not true that everything is completely dependent on the
the position of the 2nd beam relative to a properly aligned 1st beam.
Even aligning the FL4/5(3-2) aperture is much easier. After taking
the PMTs off and just looking at how things are aligned to the PMT
and filter/dichroic housings it was obvious what the problem was.
Contact me if you want more info.
tom d.
"At the heart of the slaughterhouse - always - enough room to nourish awe."
- J. Nichols
-- ============================================================================== Thomas Delohery | Internet: t-delohery@ski.mskcc.org Manager, Flow Cytometry Core Facility | Phone: (212) 639-8729 Memorial Sloan-Kettering Cancer Center | Fax: (212) 794-4019 ==============================================================================
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