GFP/Autofluorescence

A.J.Cann nna at leicester.ac.uk
Fri Nov 20 10:02:21 EST 1998


>We have just started trying to look at GFP expression in C.elegans but are
>having trouble with autoflourescence in the gut. We have tried both wt GFP
>& Clontech EGFP (red-shifted), using a BioRad confocal microscope & manual
>UV microscopes, all set up for FITC fluorescence.
>
>We would be very grateful for any suggestions/references as to how to get
>around this problem. On the other hand, we would also consider alternative
>marker genes - any suggestions?

Many thanks to all who replied. Summary of responses:

From: Oliver Hobert <hobert at molbio.mgh.harvard.edu>
There is a broad-pass excitation filter (which we routinely use in addition
to the "normal" FITC filter) that makes the gut fluorescence look more
yellowish; GFP keeps looking the same.
Here are the filter specifics: It's an "HIQ GFP ENDOW LP" filter set, sold
by Chroma (Zeiss sells Chroma filters); it's a long-pass filter (not a
broad pass as I previously said) that contains the usual 470/40 GFP
excitation filter, combined with a 535/50 Emission filter (which is the key
to see the gut fluorescence as yellowish, as this filter takes out the
red-end of the gut fluores., thus making it yellowish).

From: Thomas Burglin <burglin at ubaclu.unibas.ch>
First, get andy fire's gfp genes, they are adapted for c. elegans,
perhaps you can get them from Ian Hope in Leeds.
Second, the gut autofluorescence you can't get around.
however, if you use a normal FITC filter with a long-pass
filter (open from green to long wave-lenghts), you will find
that autofluroescence is yellow, but gfp is green.

From: Jonathan Ewbank <ewbank at ciml.univ-mrs.fr>
I'm afraid i don't have any positive suggestions to make, but can relate my
experience. when faced by the same problem, i tried a couple of mutant
strains such as CB1003 Genotype: flu-2(e1003)X.
Description: Reduced gut fluorescence, dull green.
and NG2775 (gm125) from wayne forrester in the garriga lab:
greatly reduces gut autofluorescence. I find that visualizing GFP
reporters expressed in cells in the middle of the animal is easier in a
gm125 background. gm125 is unlikely to be a known flu mutant; gm125 looks
very different from flu-2 or flu-4, the only flu1s on X. gm125 appears to
reduce the number of green or yellow spots (color depends on filters used)
from 1001s or 10001s to 10 or 20. (If I remember correctly from a previous
post, those spots appear to be some sort of storage vesicle.) Other
sources of autofluorescence, which mainly appear in older animals, are
still present. gm125 animals are reasonably healthy as homozygotes, and
males mate well.
But found that there was no significant improvement over wild type (except
that for gm125 the autofluorescence usually seen with a rhodamine filter is
completely abolished).

From: "Labrousse, Arnaud" <ALabrousse at mednet.ucla.edu>
I have two comments about the GFP/autofluorescence problem that I
encounter everyday too !
1 -  the older the worms you look at, the more autofluorescence you get.
The healthier the worms, the better.
2 - It is very well possible to distinguish GFP from autofluorescence
using a broad-pass emission filter. On our Nikon microscope, we use one
that lets all wavelengths above 520 nm to go through. That way, GFP
looks green while autofluorescence looks redish. Very recently, we
purchased an even better one, that lets every wavelengths above 500 nm
to go through. The GFP signal is stronger than with the previous filter.
In both cases, the excitation filter is the same as for FITC. I don't
remember the wavelengths in detail right now but I would be happy to
look up for you if you need them.
One final remark, though : we are using the S65T mutant GFP that has
fluorescence sprectra much closer to FITC than WT GFP. This construct is
available in the kits produced by the lab of Andy Fire
(http://elegans.swmed.edu/Worm_labs/). I never used wt GFP or the
clonetech one in worms.

--
Dr Alan J. Cann  PhD,   Department of Microbiology & Immunology,
University of Leicester,  P.O. Box 138,  Medical Sciences Building,
University Road, Leicester LE1 9HN,  UK.





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