Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi all,
I agree that knowing the color of autofluorescence is often very useful.
One might think that just looking down the eyepieces would tell us
this but, when images are dim, the cells in the fovea of our eyes are
much more sensitive to green than to other colors and our eyes can't
"integrate" dim signals over time very well.
A partial solution can be found in the color CCD cameras that are now
often found on scopes. Even consumer-grade CCD cameras have fairly
uniform color response. You can use such a camera to find the color
of your auto-fluorescence under different excitation conditions.
Images from these camera can tell some surprising tales. I recently
detected a poor barrier filter by finding some red areas in a
fluorescence image that was supposed only to be blue. With a
monochrome camera, I would have assumed that all the detected
photons were blue.
Cheers,
Jim P.
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Patricia Conrad wrote:
>
>> Has anyone had experience getting rid of autofluorescence from 293
>> cells? Two of my colleagues are trying to perform immunofluorescence on
>> 293 cells transfected with WISP3 and LRP5 respectively. However, the
>> control cells and transfected cells have high levels of
>> autofluorescence. Has anyone else experienced this with this cells
>> line? Any suggestions on how to get rid of the background staining?
>> Both colleagues see this with paraformaledehyde and methanol fixation
>> protocols, and the cells have been blocked with BSA and/or serum.
>
>Dear Patty--
>
>What color is the autofluorescence? Does it show up under all
>filters--UV through CY5? The color will give a clue to what the cause
>might be.
>
>Good luck--
>
>Martin Wessendorf
>
>--
>Martin Wessendorf, Ph.D. office: (612) 626 0145
>Assoc Prof, Dept Neuroscience lab: (612) 624 2991
>University of Minnesota Preferred FAX: (612) 624 8118
>6-145 Jackson Hall, 321 Church St. SE Dept FAX: (612) 626 5009
>Minneapolis, MN 55455 e-mail: [log in to unmask]
--
****************************************
Prof. James B. Pawley, Ph. 608-263-3147
Room 223, Zoology Research Building, FAX 608-265-5315
1117 Johnson Ave., Madison, WI, 53706 [log in to unmask]
"A scientist is not one who can answer questions but one who can
question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39
|
