GFP problems

So, my project involves looking at the dopamine neurons of C. elegans under a fluorescence microscope, but instead of seeing clearly defined neurons (like I did last year), I get something like this:

The worm above is a control worm, so it has NOT been exposed to anything. And several times, I’ve had worms that don’t fluoresce at all.
Does anyone have any idea why this is happening?

Vesicles in the gut of regular worms are somewhat autofluorescent. This autofluorescence is more severe when the worms are starved. The picture you posted looks like such autofluorescence - you can check whether that is correct by looking at the fluorescence with different filters and seeing that is is green, blue and red (not just green like GFP). If there is no fluorescence in the DA cells of the head, I suspect that the strain has lost the transgene expressing the GFP - either because it was an extra-chromosomal array or because the strain was contaminated. I use a Pdat-GFP strain and it has stably expressed GFP for several years.

Janet Duerr at Ohio University

The gut has a strong autofluorescence, particularly in older animals. If transgenic strains are left to proliferate for many months without selection, the array can become lost. If you have a frozen vial of this stock it might help to thaw a fresh sample. It often helps to pick younger worms with less gut fluorescence, keep the worms at 25C to minimize silencing of the transgene, and the gfp signal can be boosted by RNAi against RDE-2 silencing gene.

Probably, too late replying after 9 years :smiley: … but if someone else is wondering how to overcome this problem of autofluorescence masking of GFP, here is a way to do it.
DOI: 10.21769/BioProtoc.2940