Dear Fellow Microscopists,
Happy New Year to you all (not too late I hope!).
I routinely use a dilute solution of toluidine blue to quench some of the autofluorescence present in my material prepared for indirect immunofluorescence microscopy of tubulin. I am convinced that it does reduce the native fluorescence of my tissue (secondary vascular tissue of horse chestnut). However, I was floored at question time at the end of a talk at an international conference when asked, 'how does the quenching work?'. I have asked colleagues and consulted textbooks, but still do not have the answer. Can anybody help, please? [I'm sure that having the answer the question will never be asked again, but it might!]
Many thanks,
Nigel Chaffey
NIGEL.CHAFFEY@bbsrc.ac.uk

Brief rinsing with a very dilute solution of tol. blue also works to quench autofluorescence in Drosophila eyes, using thin frozen sections and FITC labelling. I'm not sure of the original rationale -the autofluorescence is very high, we were desperate for something that would work. I think just a hope that tol. blue probably binds to double bonds (??) and might just dampen things down..and so it did. Dont think I ever tried it with either rhodamine or a short-wavelength chromophore like DAPI ...does tol blue itself fluoresce under some excitation wavelengths?

Sally Stowe
Email: stowe@rsbs.anu.edu.au

Dear Nigel,
Fluorescence is the radiative decay of an excited state of a molecule to (usually) the ground state. Other decay modes exist and are always in competition with radiative decay--thus the fluorescence yield term in inten- sity formulas. Quenchers enhance non-radiative decay either by being accep- tors for energy transfer or by promoting collisional de-excitation, and by- and-large the energy transfer process is the most important. In this process, a nearby quencher molecule is converted into an excited state and the fluo- rescent molecule is converted into the ground state in a single step. The quencher then decays via a non-radiative process.
Yours,
Bill Tivol
tivol@wadsworth.org

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