Braun,
I can vouch for this. I had a similar problem a few years ago and Dr.
Haugland pointed this phenomenon out to me.
I was looking at small bacteria binding to large cells and the shift in
the flow cytometer's histogram was not at all what I expected after
seeing these highly fluorescent bacteria binding to the cells under the
microscope.
I took the small histogram shifts that and compared them to hand counts
of the bacterial binding and got very good correlation, so I stopped
worrying (so much) about it after that.
Mike
Richard Haugland wrote:
>
> The absorption of the Alexa Fluor 350 conjugate is reasonably well excited by
> the UV argon-ion laser at 351-363 nm so that is not the problem, nor is
> photobleaching.
>
> http://iw.probes.com/servlets/spectra?fileid=11045ph8
>
> However, this points out a general problem of trying to correlate fluorescence
> microscopy and flow cytometry that may be the problem here too. In microscopy
> one can sometimes visualize signals only because the fluorescence is
> concentrated in relatively small regions of the cell. In flow cytometry the
> fluorescence measured is from the entire cell. For a weak signal -- such as
> UV-excited reactive dyes -- the autofluorescence of the cell, particularly in
> the UV, can be greater than the specific signal, particularly in the UV. The
--
________________________________________________________
/ Michael J. Herron, U of MN, Dept. of Pediatrics/BMT /
/ herro001@umn.edu /
/ 612-626-4321 Mpls MN 55455 /
/_______________________________________________________/
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