Posted by
Adams,Henry P on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Quantifying-fluorescence-help-tp593618p593631.html
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalAlso, I don't think this was mentioned in the thread. When using a confocal acquire your images at 12-bit for doing 'quantitation', and aren't we really talking about doing semi-quantitation since you are comparing isolates. Quantitation infers you know what the absolute quantities of what you are measuring.
Hank Adams
Microscopy Core
Department of Genetics
U.T. M.D.Anderson Cancer Center
Houston, Tx
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Douglas Cromey
Sent: Friday, July 18, 2008 10:18 AM
To:
[hidden email]
Subject: Re: Quantifying fluorescence help
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