Complete beginner microscopist here. I was wondering if switching to an upright from an inverted confocal (both Zeiss LSM900) would offer any advantages for my particular situation. Through some miscommunication, my lab mate has cultured some samples in a polystyrene 24-well plate that we would ideally like to image at 20x. As I understand, the thick plate bottom will cause imaging issues with our current inverted scope setup because of limited working distance and light scattering. Would we get a better resolution by imaging into the top of plate (no lid) with the upright system? Thanks a bunch for any thoughts or help!
In theory yes, but I think there may be some issues with this approach:
(1) Are the wells of the plate shallow enough that you can image them with a 20x from the top?
(2) The meniscus of the culture media may create distortion.
(3) The thickness of the culture media over the sample culture can also add distortion.
You can remove most of 2,3 by removing most of the media (20x air) or adding enough to immerse the objective (20x water immersion). Also, if your 20x has a relatively low NA (less than 0.3?) you probably won’t be particularly sensitive to 2,3.
I’m doubtful you will be successful with a 24-well plate because of requirement (1) given by Hazen_Babcock. The lenses are too fat to fit inside the 24-well wells. The diameter is typically 15-16mm for those wells (see e.g. https://www.corning.com/catalog/cls/documents/drawings/LSR00003.pdf ) The depth of a well is typically 15-20mm, and the working distance for 20x will likely not be any more than 12mm. You’d have to go down to 5x to get the working distance you need. You can see the working distances and mechanical dimensions of the Zeiss lenses at:
I think it is worth trying the inverted scope: ask around for a Zeiss 20x lens that is designed for culture vessels, indicated on the Zeiss website above as “coverglass thickness” valid for ~1.0 mm. You may still get usable (though not ideal) images.