HOW TO DISCRIMINATE GFP FROM ENDOGENOUS
AUTOFLUORESCENCE - A NEW REVIEW PAPER.
Over the past few years a recurring question posted to this forum
has been, how do I see my GFP in the presence of masking
cellular or media autofluorescence ? The most frequently adopted
approach has been the optimisation of optical filters. However
autofluorescence is often intense at the very wavelength of GFP,
making simple filtering a far from idea solution.
To address this almost universal problem Nicholas Billinton and
Andrew Knight from UMIST have recently published a detailed
review article which sets out to identify the principle biochemical
sources of autofluorescence at GFP wavelengths in a wide range of
cells and species. The article then discusses a battery of
techniques that can be applied to discriminate GFP from
autofluorescence. These include spectroscopic techniques that
exploit differences between GFP and autofluorescent species in
terms of their fluorescence wavelength, bandwidth, anisotropy and
lifetime. Also chromatographic and other separation techniques,
microscopy methods and software manipulations of fluorescent
microscope images. Chemical methods are discussed including
selection of growth media and pH, and the use of coloured dyes to
quench autofluorescence. Photochemical methods covered
include photobleaching, photoconversion and FRET. Finally
biochemical considerations such as the use of more active
promoters and the choice of GFP mutant are discussed.
The reference is:
Seeing the Wood Through the Trees: A Review of Techniques for
Distinguishing Green Fluorescent Protein from Endogenous
Autofluorescence. Nicholas Billinton and Andrew W. Knight.
Analytical Biochemistry. vol. 291, April 2001. pp. 175-197.
It is hoped that this article will provide some useful insights and
solutions to the many GFP workers who struggle to see GFP in
the sea of other fluorescent compounds in the cell.
a.knight at umist.ac.uk