Hi,
I want to image live culture neurons using plastic plate instead of recommended glass plate, because they grow better on the plastic plate. We will collect Gcamp signals from mouse neurons and aim to take those images with our w-1 yokogowa confocal microscope, with 10x or 20x air objectives with PlanApo correction.
Could there be any potential issue? What are your thoughts?
Tristan Liu from Columbia Microscopy Core
Maybe? I assume they are relatively low NA objectives so they should be less sensitive to the difference in refractive index (and thickness?) between the glass and plastic plates.
The easy test, if you haven’t already done this, is to image some small (0.1um - 0.5um) fluorescent beads on the plastic plates vs glass plates to see what the issues might be.
-Hazen
Hi Tristan,
Plastic plates will be pretty limiting in terms of image quality. In addition to the differences in refractive index and thickness that Hazen mentioned, plastic plates are quite autofluorescent. The increase in background will make it difficult to detect small changes in intensity, which could be a big problem for GCaMP measurements.
The best case scenario would be to get neurons to grow happily on glass. I haven’t done it myself, but I have seen really nice healthy neurons cultured on glass. It might be worth searching the literature to see if you can find any relevant protocols to try.
Failing that, several companies now make plastic or polymer “coverslips”, marketed for samples like yours that won’t tolerate glass. I haven’t extensively tested them, and they are often not compatible with immersion oil, but might be worth a try for this experiment; they likely (hopefully!) have lower autofluorescence than standard tissue culture plates and are certainly much thinner.
Whichever plates you end up using, the most important thing is to validate your setup to make sure you can detect what you need to detect. In this case, maybe that would entail treating the neurons with well-characterized treatments change the expected GCaMP signal and making sure you can detect the changes.
Anna