Hi,
I used to strike this problem occasionally with researchers
saying the flow results were wrong because the sample was OK under the
fluorescent scope!!
To begin with these are two completely different
instruments, using different technologies. The results as far as
fluorescence is concerned will always be different.
1: The
fluorescent microscope uses a light source that emits a broad range of
light. This light is filtered into relatively broad bands for
excitation of a fluorochrome. The flow cytometer is using a laser to
generate a specific, narrow, band of light, ie 488, 633,
etc.
2: Flow cytometers and fluorescent microscopes will use
completely different filters to detect the emitted fluoresence.
(because of 1)
3: The emission from a fluorescent microscope is
finally detected with the eye, or a camera that mimics the eye. The
emission from a flow cytometer is detected with a PMT.
If you really
must do a comparison at least use a confocal microscope with similar
emission filters.
Regards
Rob W.
Original Message
--------
From: "Burke, Sean A./Sloan-Kettering Institute"
<burkes@mskcc.org>
To: "Cytometry Mailing List"
<cytometry@flowcyt.cyto.purdue.edu>
Subject: RE: signal detected
with immunofluorescence microscopy but not with cytometry
Date:
08/03/2006 06:00
I am having the
same problem! Please forward me any insights.
Sincerely,
Sean
Burke
Memorial Sloan Kettering Cancer Center
-----Original
Message-----
From: Julie Bertout [mailto:julie.bertout@ibl.fr]
Sent:
Mon 3/6/2006 3:39 AM
To: Cytometry Mailing List
Cc:
Subject: signal
detected with immunofluorescence microscopy but not
with
cytometry
Hello,
Can you explain me why sometimes I can
detect fluorescence when I
observe cells using fluorescence microscopy
but I can not detect it with
cytometry? I use different antibodies, some
work for both technique,
some don't.
Thanks,
Julie
Bertout
cytometry lab
Institut Pasteur de Lille
1 rue du professeur
Calmette
59800
Lille
France
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Received on Wed Mar 8 14:18:00 2006
