Autofluorescence suppression (Was Re: More: autofl.)

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From:"J. A. Kiernan" <> (by way of histonet)
To:histonet <>
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Here follow lots of thoughts about auto- and other
unwanted fluorescence.

On Wed, 23 Dec 1998, Karen D. Larison wrote:

> Out of curiosity, I just looked at some Bouin's-fixed lamprey muscle
>under my
> fluorescent scope and it wasn't autofluorescent ...
> ... So I don't think the picric acid in your fix is the problem.

   Spot on! This autofluorescence-reducing is a virtue of Bouin.
     The literature of fluorescence chemistry etc says the nitro
     group (present X3 in picric acid) opposes the actions of other
     arrangements of atoms (fluorophores) that make a compound

> In fact, one might worry that the picric acid would instead quench the
> fluorescence of your signal, particularly if it imparts a yellow color to
> tissue.  In general, chromogens and metals tend to quench fluorescence.

  On the nail again, but in the case of Bouin we usually make sure all
    the yellow has been washed out before staining. (Lee Luna said it
    was important to do this before embedding, and documented structural
    changes in yellow specimens stored in paraffin blocks! I haven't
    seen another publication to corroborate this. Has anyone else?
    End of long, boring aside.)

  Elements vary in their ability to suppress autofluorescence. Iron,
  iodine, mercury and osmium were all recommended in the 1950s. The
  suppressing power goes up with atomic number. My experiences with
  fluorescent methods in carbohydrate histochemistry led me ('65,'72)
  to recommend a brief exposure to dilute osmium tetroxide, followed by
  a prolonged running water wash. This removes all autofluorescence
  from all (well, I didn't try them _all_) animal tissues. Subsequently
  applied stains (fluorescent PAS-like things; labelled lectins)
  provide background-free fluorescence of basement membranes,
  secretory products etc after this minimal osmication of paraffin
  or frozen sections. The other heavy elements were less effective;
  they reduced but did not abolish the background fluorescence.
  I never tried even heavier elements, but high atomic num/wt might not
  be all that's needed. Hg (80/200) was less effective than Os (76/190)
  and both HgCl2 and OsO4 react with substances present everywhere
  in animal tissues. Perhaps I or someone else (preferably both,
  independently) should take up the search for autofluorescence-
  suppressing agents that might not interfere with immuno- and
  other fluorescent staining methods.

   It has always been possible to eliminate autofluorescence in
   published photomicrographs by controlling the exposure time
   when enlarging and printing a negative. Modern image
   processing makes it even easier to fiddle the results,
   converting the dimmer fluorescence (assumed auto-) to black
   and the brighter stuff to bright white/yellow/blue/green.
   It's now also easy to obliterate other defects in the preparation
   such as bits of fluorescent dirt and cracks and fissures
   in the section (if you were honest enough to allow a modest
   autofluorescent background, for orientation).

   Happy new year everyone!

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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