fluorescence & protein concentration

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From:<larisonk@uoneuro.uoregon.edu> (Karen Larison)
Date:Fri, 1 Oct 1999 10:56:29 +0000

Peter van de Plas asked:

Q: What about fluorescent markers. How many fluorescent molecules have to
be in close proximity of each other to generate a signal in the microscope?


This is not an easy question, particularly when you are referring to a 
fluorophore that has been covalently bound to a secondary detection reagent, 
which in turn has been used to detect an antigen located in some ill-defined 
microenvironment in the tissue.

First, it will depend on the fluorophore.  Not all fluorphores are created 
equal.  For instance, fluorescein is a particularly good fluorophore.  
However, even fluorescein has its problems (see below).

Secondly, the amino acids in the secondary reagent tend to quench the 
fluorescence of the attached fluorophore, particularly the aromatic residues.  
The amino acid residues near the site of attachment of the fluorophore will 
strongly influence the amount of fluorescence.

Thirdly, fluorophores in close proximity self-quench.  So the fluorescence of 
two fluorophores in close proximity is less than that of two isolated 
fluorophores.  If you put more than 2-3 fluorophores on an antibody, 
self-quenching becomes a real problem.

Fluorescent bleaching is also a problem.  Again, this problem varies from 
fluorophore to fluorophore.  Fluorescein is notorious for photobleaching.

The microenvironment also plays a large role in the quantity and quality of 
the fluorescent signal.  For instance, fluorescein's fluorescence is pH 
dependent.  At pH of 8 or above, fluorescein is about 10 times more 
fluorescent than at neuutral pH (don't use PBS to mount your 
fluorescein-labeled samples!).  BODIPY provides another example. This 
fluorophore is quite photostable in a lipophilic environment, but bleaches 
like crazy in an aqueous environment.  So a BODIPY-labeled secondary reagent 
is useful only if the antigen being detected is buried within a lipophilic 

Enough babble.

Karen in Oregon

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