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Quantifying fluorescence is difficult to do properly and unfortunately is
quite easy to do poorly.  At my institution I always refer questions of this
sort to Jim Pawley's excellent article (The 39 Steps: A Cautionary Tale
about "quantatative" 3D Fluorescence Microscopy).  If you can control most
of the things Jim mentions, then you are probably more talented/patient than
a lot of us.

See:
http://www.zoology.wisc.edu/faculty/Paw/pdfs/The_39_Steps_corrected.pdf

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Assistant Scientific Investigator
Dept. of Cell Biology & Anatomy, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Maria Mazzillo
Sent: Friday, July 18, 2008 6:26 AM
To: [log in to unmask]
Subject: Quantifying fluorescence help

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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