If you are just trying to compare signals from different areas in the same
image your task is much easier (as long as A/D convertor is linear) and (I
presume) independent of the "low signal" setting, so I guess this is not
what you are trying to do.  My impression is that it will be much harder to
do if gain is allowed to vary between images.  It may not be too hard if
you take all images at a constant gain, but in my work I'm always
adjusting the gain.  I assume the relationship between signal and gain is
not linear so if gain is allowed to vary, a standard curve will have to be
done not only for different intensities at a single gain (which I expect may
be linear but this needs to be shown), but also for numerous gain
settings.  Of course zoom will need to be constant, too.  Whether or not
you will be comparing different specimens, your various intensity
standards will probably be on different slides (otherwise you will have to
be sure you know which is which), so you should show how
reproducible the signal is for a given intensity bead prepared on multiple
slides, all imaged under identical settings.  If it is significant, do it for
multiple settings and several intensities of bead.  This will let you know
how much noise there is in addition to that due to electronics.  Could be a
source of problems.  If your dyes bleach but your standards don't, then
establishing a standard curve will be irrelevant to your samples unless
photobleaching is minimized during imaging of specimens.

 I have thought about it but never tried it.  Please let me know your results.
 (My experience is with a 1024, I assume a 600 is not too different.)

Richard
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>>> Wayne Arnold <[log in to unmask]> 09/04/97 08:29am >>>
 Our goal is to try and come up with a reasonable
estimate of the  relative amount of a particular antigen
present in different areas of the cell based on different
intensities. I have been advised that the A/D
converter is linear but that I should be careful not to set
the blacklevel artificially high. That been said I am trying
to prepare a calbration curve for the Biorad MRC 600 to back
up the theory.

I have tried Molecular Probes new  intensity calbration kit
for confocal microscopes. Has anyone used this and would
they have a proper procedure for doing the calibration ?
When I scanned the standards, I had to have the enhance
button  set to "off". However I routinely scan samples at
"LOW SIG" and I have been told by Biorad that there is not a
linear relationship between the 2 settings. Therefore, if I
try to get some standards custom made I'm not sure what
intensity range to ask for  to be in the range of the "LOW
SIG" setting.Does anyone have an alternate source for some
standards? Has anyone tried this themselves?