At 05:06 PM 1/6/98 -0800, you wrote:
>Hi There!
>        Does anybody know about hemoglobin auto-fluorescence? I have red
>blood cells showing up as non-specific background in my Fluorescence In
>Situ Hybridization slides. They show up in PMT1 and 3, when excited with
>either all lines, or 568, or 647 alone. PMT 2 is clean. Any ideas?
>
>
>Kathy
>
>
>Kathy Spencer
>Scientific Associate
>The Scripps Research Institute
>La Jolla, CA  92037
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>

Hello Kathy,
I am not sure about which wavelength range of fluorescence each PMT in your
microscope is measuring. I can assure you with full confidence that native
hemoglobin, with a Fe-heme prosthetic group in each subunit, will not
autofluoresce. The only fluorescent groups in hemoglobin are aromatic amino
acids which are excited in the UV region (below 340nm).  On the other hand,
if the red cells you are looking at contain free porphyrins (this occurs in
certain pathological conditions) or Zn-protoporphyrin (common in lead
poisoning) you may get some fluorescence. A few years ago, I published a
couple of papers describing the quantitative determination of the
distribution of the cellular concentration of these compounds in red blood
cells by image-based cytometry. If you are really interested in knowing
what is the source of the signal, I suggest you try measuring the
fluorescence spectrum of lysed cells or you may extract the porphyrins and
measure the fluorescence of the extract to determine if indeed the red
cells you are looking at have a high porphyrin content.  If you wish to
proceed this way, you are welcome to get in touch with me and I will give
you references to methods to do this.
Sincerely,
_____________________________________________________
Massimo Sassaroli, D.Sc.
Department of Physiology and Biophysics
Box 1218
Mount Sinai School of Medicine
One Gustave L. Levy Place
New York, NY 10029-6574

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