Reply to:   RE>Measuring relative fluorescence intensities
Hi Kem,
  Intensity calibration: Check out the new "MultiSpeck(TM)" multispectral
fluorescence intensity standards beads now available from Molecular Probes
(MP). They are described on page 6 of MP's BioProbes #18 (see also back cover).
They are arranged in a monolayer of 4 um beads of low, medium, and bright
intensities (the packaging should include intensity quantitation). The dim
beads are very dim fluorescence (may not be visible without dark adapting your
eyes) but no problem for the 12-bit cooled CCD I used. The beads fluoresce at
many different wavelengths. I'm predicting they are going to be great for
intensity calibration and demonstration of registration of images acquired at
differentwavelengths (or lack thereof). MP should have the beads at their booth
at ASCB.
  I have no opinion on either the optimum fluorochrome or confocal vs.
conventional microscope, other than to say that you should use the best oil
immersion objective possible with your culture chamber (examples: 40x 1.3 NA or
60x 1.3 NA Fluorite).
George McNamara
Universal Imaging Corporation
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--------------------------------------
Date: 12/6/93 17:22
Subject:      Measuring relative fluorescence
From: "k.a. rogers" <[log in to unmask]>
To: Multiple recipients of list CONFOCAL <[log in to unmask]>
 
        How can I best determine if the "amount" of labelling of two cells
differs quantitatively.  Would something like internal flourescent beads
work?  I am particularly interested in the amount of cell surface protein
on cells at different times in culture.  Which fluorochrome is best to
use, or does it matter?  Is a confocal microscope a good instrument for
such work.
 
                        I would appreciate any suggestions.
 
                                        Kem Rogers
                                        University of Western Ontario