Hemoglobin fluorescence

Dave Bates dobates at ucdavis.edu
Thu Mar 23 14:30:09 EST 1995


In article <3544 at alsys1.aecom.yu.edu>
gottfrie at alsys1.aecom.yu.edu (David S. Gottfried) writes:

> In article <3kqd92$np1 at mark.ucdavis.edu>
> dobates at ucdavis.edu (Dave Bates) writes:
> 
> > Does anyone have a protocol for stopping hemoglobin fluorescing? I'm
> > finding it a smidgeon difficult to do fluorescence antibody work on my
> > blood vessels (well not actually my blood vessels - ouch), when if
> > there's any red cells left in it wipes out anything I was looking for.
> > Not to mention the poor wee soul in the next door lab who's looking at
> > immunohistochemistry of the red cells themselves!
> 
> What is the wavelength that you are exciting at?  Hb will only
> fluoresce, and then only VERY weakly, when excited in the
> tryptophan/tyrosine region (<300 nm).  If your fluorescent antibody has
> an absorption to the red of that, then you are not seeing Hb
> fluorescence.
> 
> One somewhat sophisticated method to eliminate the Hb fluorescence is
> to use time-gating to observe only fluorescence that is emitted >1 ns
> after the excitation source.  Since 98% of the Hb fluorescence is very
> short lived (25 ps lifetime - see references of Bucci et al.), this
> will be unobserved.
> 
> Good luck,

Ok maybe it's not the hemoglobin, but the red cells do fluoresce
strongly when fixed with formaldehyde under FITC fluorescence
conditions (450-490 ex, 510-530 em). They do, honest. It's not just me
either. They look green. Really. They do. Bright green. and Fuzzy. With
caterpillars coming out. And the toadstools made me do it. Oh no I
can't stand it any more, heeeelp



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Dave Bates PhD                                     Tel: 916 752-7081
Postdoctoral Researcher                            Fax: 916 758-2554 
Dept of Human Physiology                         email:
dobates at ucdavis.edu
University of California at Davis                drink: anything
Davis, California 95616, USA            No I don't have a sense of
humour 
                                                now buy me a beer      
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