We are using a Bio-Rad MRC 600 Confocal.

I have been told I can safely say that if I have an image where the
intensity of a signal in one subcellular compartment relative to another
(in the same image) is 4 fold (e.g.. pixel values averaging 50 and 200),
then the corresponding protein (to which our antibody is directed)
concentration should be 4 times higher in one compartment relative to
the other (assuming accessibility etc. are equal).

We must also make sure the black level is set up properly to prevent
artificially cutting off the bottom. For e.g.. when set properly we may
get two averages of 50 and 200 in two compartments (4 fold difference).
If set too high (or is it low?) we may for e.g.. cut off the 0-40 range
leaving us with values of 10 and 110 (11 fold difference).

Others have said that even with raw images there are just too many
variables in microscopy to be able to make such an assumption.


Biorad has told us that the relationship between two intensity values at
a given setting in an image should be linear.

We then set out to prepare standards to prove this to ourselves. We want
to prepare our standards to be able to get readings within the range of
settings we are using (gain 500-600, enhance set to low signal, ND
filter 2 and using the k1/k2 filter blocks for texas red and fitc). We
tried the Molecular Probes standards however they are much too intense
and we have to use the set the enhance setting to off. We also tried to
dilute a secondary conjugated to texas red and serially dilute this. We
then placed a drop on a slide and placed a coverslip over it. However
even in an undiluted form we had to up the gain to near the top.

Does anyone have any comments or suggestions for a source of standards
as well as on the whole issue of the relationship between intensities
(WITHIN AN IMAGE).