You should be able to get the information you want by using your
original experimental setup with the Molecular Probes standards, but
with one change: decreasing the excitation intensity, either with a
stronger ND filter, an additional ND filter, and/or turning down the
laser power (if you can), .

Jeff Reece
NIEHS

> ----------
> From:         Wayne[SMTP:[log in to unmask]]
> Sent:         Wednesday, April 29, 1998 10:41AM
> To:   [log in to unmask]
> Subject:      intensity relationships within an image
>
> 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).
>