C. elegans are a historically relevant model for many biological and chemical laboratories. Microscopy with C. elegans has been explored previously, and our lab focuses on the use of fluorescent microscopy with live nematodes. Imaging live C. elegans raises several issues such as how to treat the samples, how to immobilize the subject, and how to quantify the fluorescence. Previous studies imaging C. elegans mostly focus on fixing the worms with chemicals and do not allow for live imaging. Other studies have used a slide and coverslip method for immobilization with minimal sample volume or the addition of agarose pads to the slide. In our experience, the C. elegans move too fast for fluorescent imaging or their bodies are crushed under minimal volume conditions. Ethanol is a known locomotion inhibitor for C. elegans at concentrations near 400mM (McIntire, 2010) and it seemed to be a likely candidate for immobilizing live nematodes. Another advantage of this technique is that it allows for the recovery of live nematodes.
C. elegans are grown as plate culture at 19 oC for 6-7 days, and then synchronized for a uniform developmental stage harvest as described in Wormbook (Stiernagle, 2006). Plates are treated with fluorescent dyes in M9 buffer in the presence of E. coli for 20 minutes. C. elegans are removed from the plates and 400 mM ethanol is added to the solution for 5 minutes to anesthetize nematodes. A five minute treatment with 400 mM ethanol immobilizes C. elegans for up to twenty minutes of imaging. From each sample, 10 µL of the solution is pipeted onto a Thermo Scientific ColorFrost Plus Slide™ and covered with a Columbia Calibre® glass coverslip. The C. elegans are imaged with an Olympus inverted microscope IX81/DP71, using appropriate fluorescence filters. To obtain quantitative microscopy results, images are analyzed for pixel intensity using ImageJ Software provided by NIH.