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Hi Maria,

The main issue you will face is photobleaching.  Standardising the
exposure times and the use of calibrated beads will help.   They will
give you relative values where you can at least get a relative but not
absolute result but you will need to be very disciplined and methodical.

Wide field and point scanning confocals have very high bleaching rates.
I would also like to reinforce Julio and Joel's remarks about staying
away from saturation of your fluorophores in excitation. The spinning
disc confocals have vastly lower bleaching rates, at least one order of
magnitude less and because of this your relative determinations should
be better. Look for a system based around the Yokogawa unit say from
Andor of Perkinelmer.  These systems are likely to give you the best
results in my opinion.

As pointed out in other responses there may be some issue with the
different emission spectra of the fluorophores you are investigating and
the InSpeck beads if you use these.  However, if you are not observing
and spectral shifts and you are not at saturation in excitation then
these differences should be acceptable and the proportionality should
stay the same.

If these were my experiments I would set up a good positive control with
an standard off the shelf fluorescent probe to ensure everything is
running smoothly with each experiment.

I hope this helps.

Cheers

Steve Hunter

-----Original Message-----
From: Maria Mazzillo [mailto:[log in to unmask]] 
Sent: Friday, 18 July 2008 11:26 PM
To: [log in to unmask]
Subject: Quantifying fluorescence help

Search the CONFOCAL archive at
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I am a PhD student at Auburn University just getting started into my
thesis work.  I am 
looking for a reliable method for quantifying fluorescence.  

My project deals with marine dinoflagellates (zooxanthellae) that reside
intracellularly in 
hosts (usually cnidarians).  I am working with cultures or isolates of
only the dinoflagellates 
for my confocal work.  I have an antibody that was created against the
surface secretions 
of mucilage (secreted as part of a daily cycle by the alga) from one
strain of zooxanthellae.  
I am attempting to use this antibody to label various strains to
identify differences in 
mucilage between them.  Thus far, I have seen that the strain the
antibody was created 
against labels around the cell fairly brightly.  Most samples either
show this or a complete 
lack of labeling.  However, a few samples show a faint fluorescence
lifted off the cell 
surface.  I would like to be able to quantify this fluorescence in
comparison to either the 
control strain that the antibody was created against or against a known
fluorescence.  

Any help on ideas for this, or places to look for ideas would be greatly
appreciated.

Thanks!

Maria Mazzillo