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Dear Jean and others,
if the reviewers presume there is anything like a linear relationship
between the intensity value measured from the image and the
concentration of molecules in the sample, this could be an interesting
paper for them to read:
Biophysical Journal Volume 92 April 2007 2926-2943
Deconvolving Single-Molecule Intensity Distributions for Quantitative
Microscopy Measurements
Sarah A. Mutch,* Bryant S. Fujimoto,* Christopher L. Kuyper,* Jason S.
Kuo,* Sandra M. Bajjalieh,y
and Daniel T. Chiu*
Departments of *Chemistry and yPharmacology, University of Washington,
Seattle, Washington 98195
regards, jens
---
Dr. Jens Rietdorf
Head Microscopy
Novartis Research Foundation
Friedrich-Miescher-Institute, wro1066.2.32
Maulbeerstr.66, CH-4058 Basel, Switzerland
phone +41(61)69-75172 mobil +41 798284737
Email:[log in to unmask]
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Jean Brennan
Sent: Dienstag, 26. Juni 2007 02:00
To: [log in to unmask]
Subject: Re: Quantitative immunofluorescence
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I have several scientists that want to quantitate intensity of eGFP
expression. We pfa perfuse, post fix for 1hr, cryoprotect in 30%
sucrose overnight, cut, warm the slide, hydrate in PBS, mount with
Prolong G and image on Zeiss 510 Meta. For controls we use WT tissue
(all mouse brain). I set the upper and lower limit based on the samples
I have that day and take all groups being compared in the same confocal
session. I'm not very comfortable with this but they are insistant
about the results. Any comments or suggestions?
Jean Brennan
Research Specialist
Rothstein lab, Dept of Neurology
Johns Hopkins University
600 N. Wolfe St., Meyer 6-174
Baltimore, MD 21287
410-614-4119; FAX: 410-955-0672
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>>> Jennifer Waters <[log in to unmask]> 06/25/07 2:57 PM >>>
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There are many potential problems with quantitating intensity values of
images of immunofluorescently labeled specimens as a means to determine
the relative amount of the protein that was
labeled...extraction/fixation, antibody penetration, nonspecific
binding, conditional epitopes, differential binding of a multivalent
probe, etc. Nonetheless, this is technique is used and published all of
the time, often with very few controls. I had a discussion with a user
this morning who had 2 out of 3 journal reviewers insist he quantitate
the intensity of a soluble protein using immunofluoresence.
Thoughts? Anyone have any favorite references on topic? I just enjoyed
reading Melan and Sluder, J. Cell Sci 101, 731-743.
--
Jennifer Waters, Ph.D.
Director, Nikon Imaging Center at Harvard Medical School
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