Hi Stephen,

I have a few questions for you about your calibration problem.
I use an MRC-600 so I'm assuming the photon counting mode is the
same with the 1000.

(1)  What is the range of greylevels you are working with?  You mentioned
that it is not the full 8-bit range but what is it exactly?  Are you able
to use the same range to calibrate (with the InSpeck beads)?

(2)Can the reduced sensitvity - not in detection but in quantification -
of the photon counting mode be a problem?  e.g.  Say the signal from
the beads ranges from 0 - 50 greylevels and you create 9 bins using photon
counting.  You have effectively reduced your dynamic range from 51 to 9
greylevels and the quantization may mask the actual diffrences in
intensity (I) among the beads.  I'm sure there is some range of I among
the beads in a particular calibration sample and if this range is
close to the number of greylevels in each of your bins OR if the error
is biased in one direction (most of the intensities are higher than
the mean in one sample and most are lower than the mean in the next),
the quantization may amplify the error.

(3)  What type of curve do you get with the beads?  Is it exponential
or does it simply max out before you expect it to?

-Tracy


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Tracy Richmond McKnight                 email:  [log in to unmask]
Department of Human Physiology          TEL:    (916) 752-5584
School of Medicine, MS-1A               FAX:    (916) 752-5423
University of California
Davis, CA 95616
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On Mon, 20 Jan 1997, Stephen T. Haley, II wrote:

> 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
>