Hemoglobin fluorescence

David S. Gottfried gottfrie at alsys1.aecom.yu.edu
Thu Mar 23 09:56:21 EST 1995


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,
***************************************
David S. Gottfried
Department of Physiology and Biophysics
Albert Einstein College of Medicine
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