pH, fluorescein and internalization.

Friends,

Following the discussion on receptor internalization, I would like to share with you a couple of questions, and hope you can help with clarifications. 

I can understand that fluorescein can be used based on its pH dependence as an EVIDENCE of internalization, by detecting the loss of fluorescence (by flow or microscopy) due to acidification of endosomes. But certainly it cannot be used as a MEASURE of internalization, or can it? how can you control or detect the pH of the vesicles given your treatment, the time elapsed, the asynchronous nature of the process, the continuation of the metabolism of the endocytic pathway, etc? For measuring it would have to be used together with a non-pH sensitive probe, as Thomas D. mentioned. And still then I think some problems would appear regarding the determination of the internalized vs non-internalized fraction.
I understand fluorescein is used as a tracer or marker of the endocytic or transport pathways in a variety of ways but wouldn't it be a bad choice given the expected loss of fluorescence? Mol probes has an interesting twist, where highly conjugated proteins or dextran cause internal quenching and after proteolysis there is dequenching, but once again the pH would affect fluorescein and it would be a bad choice given non-pH dependent alternatives. (http://probes.invitrogen.com/handbook/sections/1407.html). 
Finally, applications where fluorescein is not used in the endocytic pathway (like the FDA viability assay) should not suffer from these problems, or maybe it would be more cautious to say that they probably suffer to a much lesser extent from these uncertainties.

Therefore, given fluorescein's subtleties and given the availability of other probes which overcome them, what escapes me, as I said in a previous post, is: why is it still so widely used? Am I missing something? or is it plain lack of knowledge regarding fluorescenin's pH dependence?  

Thanks,

uriel.
Uriel Trahtemberg, M.Sc.
MD/PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL 

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