Dear,

I should say NO.
There is the linearity problem of the detetcor (cfr previous discussion)
There is the problem of deviation from Lambert-Beer law at high
intensity. Saturation is one cause for this.
Than there is the geometry of the illumination system.
A good discussion of this was formulated by your compatriot: Rigler.R. in
1966. Acta physiological Scandinavica vol 67 supplementum 267.
If one stains pigeon erythrocytes with acriflavine-Feulgen or
pararosaniline Feulgen at different concentrations and after different
periods of hydrolysis one can obtain a stable set of nuclei displaying
absorbances ranging from .01 to 1.5. These objects than can be used to
look for fluorescence. It is found that at higher dye concentrations,
the fluorescence intensity does not level off but decrease due to
reabsorption and inner filter effects. This is in confocal as well as in 
conventional cytometry. 
We can refer to:
Prenna G.et al: Histochemical Journal 6 p259-278 (1974)
Prenna et al. Histochemical Journal 6 p467-489 (1974)
Personaly we also have seen this 
Cfr. Tanke et al. Cytometry 2 p359-369 (1982)
and Van Oostveldt en Bauwens J.Microscopy 158 p121-132

The fact is, contrary to cuvet fluorometry, in microscopy you have
epiillumination (which levels of at high fluorochrome concentration) and 
oblique illuminations, which results in a decrease of measured
fluorescence at high fluorochrome concentrations. 

To make things short: illumination geometry, defined by numerical
aperture, influence the balance between real epiilumination and oblique
illumination and detections, and therefore the aperture of the object
will influence fluorescence/concentration relationship at high
fluorochrome concentrations. In this case high is absorbances above 0.5.

Probably this sound old, but I think this litterature has to take into
account, because physics are not changed by the introduction of a
confocal microscope. They are just proved to be valid.

Greetings,


Patrick

On Thu, 30 Apr 1998,
Annika Altskar wrote:

> We have a question related to intensity relationship within the
> image.
> We are working with a polysaccharide labelled with RITC.
> Is the intensity response from the dye linear to the concentration of
> the stained material in the whole concentration interval of the dye?
> 
> Annika Altskär
> SIK The Swedish Institute for Food and Biotechnology
> 

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Patrick Van Oostveldt
Lab. Biochemistry & Molecular Cytology
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