Hello everyone,

I am working on a project to conduct calcium imaging in the neurons of live immobilized nematodes (C. elegans) in response to a thermal stimulus (changing temperature, i.e., 15C → 30C). I’ve seen these methods work in other studies, but unfortunately, I haven’t been able to replicate them. Specifically, when analyzing the data for my positive controls (C. elegans expressing GCaMP in the AFD neurons), it looks like the neurons do not respond at all to the temperature change. We are not sure if the lack of response is due to (A) the nematodes simply do not respond to these temperature conditions (which seems unlikely based on the literature), or (B) our equipment and/or analysis is not suitable or powerful enough to detect responses. So far I have tried adjusting many of the parameters with no success. I was wondering if anyone here has any experience with thermal calcium imaging and/or technical expertise, and would please advise. I would greatly appreciate it!

The equipment we are using:

• Microscope: Olympus BX53
• Fluorescence light source: Olympus U-HGLGPS
• Camera: Tucsen FL 20BW
• Capturing images using Mosaic v2.3 software (from Tucsen)
• To apply thermal stimulus: Custom thermal stimulus rig (used in

  Article The neural basis of heat seeking in a human-infective parasitic worm

  )
• The neurons express GCaMP7b calcium indicator
• For analysis, I have been using ImageJ/FIJI, using the ROI manager to circle the neurons & track mean pixel values per frame in the video/image stack (.avi files). (Similar to the method described here: akiramuto.net/archives/148)
• Calculating ΔF/F = (F-F0)/F0

I have tried capturing images with these conditions:

• 100ms exposure time, 8fps, resolution 5472x3648
• 100ms exposure time, 10fps, resolution 1824x1216 (3x3 bin, high speed)
• 50ms exposure time, 19.6 - 19.8fps, resolution 1824x1216 (3x3 bin, high speed)

I thought lowering the exposure time and/or increasing the fps could potentially better capture the fast calcium transients of GCaMP7b that may have gone undetected at a lower fps, but this didn’t seem to be the case.

I have also tried administering different temperature ramps and different rates of temperature change (C/s), but all resulted in no response.

Does anybody have any ideas of either (A) what could be the problem or (B) any other methods I could try to fix the problem?

Please & Thank you!