Increasing Z-stack range


I am trying to perform confocal 20X Z-stacks for the entire z-axis of 2-3mm thick, coronal adult mouse brain slices using 85% glycerol as mounting solution. I have been using a blutack to rest a coverslip on a slide with the slice. I have never had trouble with hydrogel embedding using a dessicator and mineral oil, or clearing using a hybridization oven and daily washes.

Normally, I can only see approximately 800um deep into the slice if I don't preincubate the section in glycerol (the objective lens can be lowered further, but it doesn't give a good signal). The other day, I left a slice in a 15mL tube containing glycerol for about 36 hours, and managed to get 2000um deep (I am using a fancy objective lens designed for SCALE meant to have a large working distance).

Does anyone know of factors or methods which increase or decrease the depth to which one can image in thick sections of cleared tissue?



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    Have you tried adjusting the collar on the objective at deeper depths to compensate for spherical aberration?
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    I think you figured one method out already - it will definitely help to incubate the samples for several hours or overnight in whatever solution you are using to image. It takes time for the PBST, which is not an ideal refractive index match for imaging, to diffuse out of the sample and be replaced by the better matched imaging solution.
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