Visualizing weak fluorescence in multicellular stages

Contributed by Harry MacWilliams, based on a procedure developed by Kees Weijer

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This technique optimizes the visualization of fluorescence and is especially useful to work with constructs with labile versions of GFP.

Grow cells in low-flourescence medium as decribed here.

Set up the strains to develop on a thin layer of low-fluorescence agar. The first choice is "Noble agar", which is sold by most suppliers. If this is not available, one can also use agarose, the same as is used for DNA gels, or one can clean up regular bacteriological agar by stirring it in cold water for 10 minutes and letting the agar grains settle out, repeating the procedure several times until the supernatant no longer has detectable fluorescence (a fluorimeter is helpful here; it doesnt have to be a good one).

After slugs develop, cut out small squares of agar, bearing the slugs, and invert these into a drop of silicone oil (best) or water (also works) on a large cover slip. Note that the side of the agar square bearing the slugs is DOWN, and the slugs are now in oil, between the agar and the cover slip. Note also that there is no microscope slide in this prep; one only needs a cover slip.

Place the cover slip, bearing the slugs, on the stage of a cell culture microscope (inverted microscope, objective is underneath). The agar block is on the top and one looks from beneath, through the cover slip, at the slugs. Examine with a 20x-60x objective, using fluorescein filters. These filters will work for both wildtype and s65t gfp, better with the latter. Use oil immersion objectives if available.

With a decent camera, the weak autofluorescence of Dicty tissue is easily visible. Prespore cells and differentiating spores have stronger autofluorescence, and it is important not to mistake this for a GFP signal. You should thus have aggregates of untransformed cells available as controls. If your signal is only slightly above autofluorescence, it can be helpful to perform an autofluorescence subtraction. This is relatively easy, provided that your GFP is not UV excitable (for instance, s65tgfp). Make images of one of your control preps using both DAPI and fluorescein filters. Using ImageJ or a similar program, subtract the background from both images, then determine the ratio of the flourescein to the DAPI signal in the resulting pictures. Next, make DAPI and fluorescein images of your GFP-expressing tissue. Again, subtract background from both images, then multiply the DAPI image by the image ratio determined above from control cells. Subtract the resulting calculated autofluorescence image from the fluorescein image of your GFP prep.

These preparations are stable for a few minutes, sufficient for photographing, but should not be left standing around for hours, as 1) the slugs become anoxic and develop multiple tips; 2) the preparation eventually will dry out and the slugs will get squished.

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