Hello readers,

        Excuse me. I am not a listed member of this group
but I would like to ask a question to readers of this thread.

        Doesn't Stephen need to work out his linearity
directly with samples rather than relying on microbeads?

        The imaging system can be non-linear but it can be used
within a linear range by adjusting an input as well as
controlling the gain and the offset of the imaging system.
A valid linearity can be obtained only if
the output shows a linear response to the change of the input
implemented by, for example, changing the primary concentration,
or changing the FITC concentration in his samples.

        If Stephen's samples were prepared within the linear
range of input parameters as mentioned above and this input was also
within the linear range of the imaging system, then, his observation was
valid enough to quantify relative fluorescence.

Best regards,

Desok Kim

In article <[log in to unmask]>,
   "Stephen T. Haley, II" <[log in to unmask]> 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

Desok Kim, PhD                  Phone:919-966-9259
Burnett Womack 460, CB#7235     Fax  :919-966-5722
Dept. Surgery (Urology)         email:[log in to unmask]
The Univ. of North Carolina
Chapel Hill, NC 27599-7235, USA