Coverslip Secrets
Coverslips typically come in 4 thicknesses: #0 = approximately 0.10 mm thick #1 = approximately 0.12- 0.15 mm thick #1-1/2 = approximately 0.17 mm thick #2 = approximately 0.2mm thick. For the best image, you want the right coverslip: 170 microns. However, in the case where you want additional depth penetration into your sample, you might use a thinner coverslip, and trade optical quality for increased depth. This is because each objective has a maximum working distance at which it can focus. The thinner the glass, the deeper you can ultimately focus.
For example, our 40X/1.30 Plan Neofluar can penetrate 120 microns into your sample if you use the #1.5 (170 microns thick), but you could go 180 microns with a #1 (which is 90-110 microns thick), or 230 microns using a #0 (50-60 microns thick). Image quality will degrade at all depths with these thinner glasses, but you'll be able to image deeper into the sample.
Since these estimates assume an optically clear sample with no refraction, practical distances will be somewhat less than that, although you could approach these distances by employing clearing steps (with e.g. methyl salicylate) and a mounting medium with the proper index of refraction. Also note that the thickness values are nominal, and can vary by large amounts, but the general message is still the same.
All of the foregoing assumes that you've already taken care of the most common problem we encounter: Do not allow excessive distance from your coverslip to your sample. This can not be stressed too highly. Some people worry about crushing their cells. In practice, it's usually the opposite problem; if the cells have a 100 micron layer of medium between them and the coverslip, image quality will suffer greatly, unless you pay a lot of attention to the optical quality of the medium. The ideal situation is to grow the cells on the coverslip that you will be imaging through, reducing the distance to zero, and giving us the clearest possible image. This is why we frequently recommend Mat-Tek dishes .
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