Hello List Members,
     I have already sent this to the capable people at Bio-Rad but
thought I may post it to the list for some feedback.  Thanks in
advance for any ideas.

     I am trying to do some "relative" quantitative immunoflourescence

on FITC labelled tissue sections using a Bio-Rad MRC-1000.
I was happily collecting data.
The levels of the surface markers I am trying to measure went up or
down as I expected. The trouble arose when I tried to check the
validity of my measurements with some InSpeck beads from Molecular
Probes.  The beads are calibrated to several relative intensities. To
make a long story short I found out that the system is not linear. I
am working in the lower portion of the 0-255 scale. The laser settings
Iam using are as follows in Photon counting mode: Iris=3.0,
Gain=1250,Multiplier=6, and the Laser set to 0.3, 1% or 3%, depending
on the relative brightness of the bead. I have played around with the
recommended zoom settings (from Pawley's handbook) to satisfy the
Nyquist criterion but it does not seem to help.

Thanks in Advance for your suggestions,


Stephen T. Haley
Graduate Student
University of South Carolina School of Medicine
Department of Microbiology and Immunology