[Histonet] Coverslipping for excellent optical quality

[Jonathan Cremer]

Dear Histonetters,

I am currently reading up on proper use and maintenance of microscopes, and inevitably stumbled across Köhler's illumination. No one ever explained this to me, although I have a 'microscopy protocol' that comes close to properly explaining the correct setup.
So now that I know how to properly use a microscope fitted with such a system, there is still the issue of the coverslip/mounting medium which are considered the 'first lens'. I understand why there is a narrow tolerance for coverslip/mounting medium thickness to achieve optimal performance, but how do you control that thickness?

Currently, when I mount a slide, I keep it wetted with xylene, put a sufficient amount of mounting medium on the coverslip and mount in the usual 'rolling' motion. I then push on the coverslip to remove trapped bubbles (if any) and be sure to spread the glue under the entire coverslip.
However, how can one be sure about the thickness of the mounting medium layer? Using more/less glue and applying more/less pressure to the coverslip must surely influence this? Especially since we're talking microns.

Does anybody have any insights or tips, or I am just nitpicking and trying to achieve an impossible result? :)

Regards,
Jonathan

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Jonathan Cremer
Laboratory Technician
Gastro-enterologie
KU Leuven, Belgium