I hope I'm not offering misleading information, but Chapter2, Fig. 8 of
the new edition of Pawley's handbook gives a graph of the response of the
MRC-600, MRC-1000, and MRC-1024 in fast photon-counting mode. They all
show nonlinearity beginning at low counts/pixel as you describe.

Are the measurements that are being compared all from equivalent ND
filter settings? What is on my mind here is the saturation of the
fluorochrome by the higher intensity excitation as you get to 3%
transmission.

Sincerely,

Dale A. Callaham
Central Microscopy Facility
University of Massachusetts
Amherst, MA 01003 USA

email: [log in to unmask]


>
> This is consistent with our findings on an MRC-600.
>
> ________________________________________________________________________________
>
>
> Paul Goodwin
> Image Analysis Lab
> FHCRC, Seattle, WA
>
> 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
> >
>