Hi Stephen and Chip,

> > 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.
>
> Hi Stephen,
> more likely site of problem is in your setting of the background in your
> image. If you take an image where there is no fluorescence (air, water,
> whatever), and then make a histogram of the results, all your values in
> the image must STILL be above zero. Otherwise when you go to take
> fluorescence images, you will not be linear.

That's the most probable cause. I don't have any experience with the
MRC1000, but in the MRC600 a black level of 5.00 used to be the correct
one for extrapolating "no fluorescence" to 0.

However, in the MRC600 I found that the best way of checking this was
to use the transmission detector at different laser intensities
(making sure that the collected images are not saturated).
I had an A/D conversion card for on-line power control of the Kr/Ar
laser so displayed intensity vs. laser intensity plots were really easy.

Without laser power control, probably the best way to check this
would be to make a very accurate measurement of the transmittances of
the ND filters and then use the transmission detector to measure
displayed intensity vs. laser intensity at different black levels.
As I said, in the MRC600 a black level of 5.00 would extrapolate to 0.

Hope this helps

Carlos