Posted by
cromey on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Quantifying-fluorescence-help-tp593618p593629.html
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalQuantifying 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.pdfDoug
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
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
office: AHSC 4212 email:
[hidden email]
voice: 520-626-2824 fax: 520-626-2097
http://swehsc.pharmacy.arizona.edu/exppath/Home of: "Microscopy and Imaging Resources on the WWW"
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On
Behalf Of Maria Mazzillo
Sent: Friday, July 18, 2008 6:26 AM
To:
[hidden email]
Subject: Quantifying fluorescence help
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalI 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