MicrobeHunter.com Microscopy Forum

So 0.17mm is the expected coverslip thickness of standard objectives, and there are articles saying the coverslip has to be 17mm, and it is particularly important with increasing NA, so coverslip size #1.5 has to be used, I understand that. However, looking at various suppliers I see #1 is the default size and many places even do not carry #1.5 in stock, and they also say #1 plus the thickness of mounting media makes it 17mm. So I am confused. Why is size #1 so popular ? Does the magnification used in most (clinical?) environments not require #1.5 or is it too expensive to make #1.5 comparing to #1 ?

Mete

Hi Mete,
what is expensive is limiting the tolerances or selecting to stay within narrow tolerances. Usual quality cover slips have a tolerance of +/- a few hundreds of a mm. Zeiss offers cover slips with a thickness of 0,17 +/- 0,005mm, a lot tighter tolerance and much more expensive.
The dominance of #1 cover slips comes from the fact that mountant adds to the cover slip thickness. If it were of the same refractive index the calculation would be simple: 0,14mm cover slip + 0,02mm mountant = 0,16mm. With mountants of other thickness the effect will go in the same direction but be more difficult to calculate.
I always try to make my preparations upside down so the object has the best chance to lie close to the cover slip.
In case of oil immersion objectives the cover slip thickness is less important - thicker glass, thinner oil layer.

Bob

I guess then it makes sense to use #1.5 if the sample is prepared very good (thin etc.), but for general use #1 sounds like a better option. I had probably the wrong impression that #1.5 is always better.

Yeah to MB's point #1 will be ideal for your wet mounts of small pond life or your veterinary fecal samples or really anything routinely and quickly prepared for quick examination for a specific purpose better have a slip too thin and lose a tiny bit of resolution hardly any at all in your achromats than to have one too thick and not be able to focus at all. There's no vet tech out there that wants to mess around with a .95 40x apo anyway barely see any depth if you get the thing working that is.

Prepping a permanent slide for research purposes or just to see the finest structures you better pull out your micrometer and measure.

Thin slips also work if your subject is not entirely flat and you want to focus on a structure in the interior.

OK I understand, thanks

Last but not least, #1.5 are less fragile than #1 - especially when handling them for cleaning.

I was just thinking the amount of mounting media to be used. Assuming its RI is similar to glass, and lets say I am using #1 and it is 0.15mm, and the sample is on the slide, and coverslip is 22x22, assuming sample volume is negligible, 22x22x0.02 ~ 10mm3 so 10uL. That assumption a drop is roughly 50 uL is way too much it seems, which is also my experience with eye droppers and micropipettes.

The drop is more than needed to fill a 0,02mm gap, but it doesn't harm if there is mountant below the object. When I make e.g. a diatom strew slide I place a drop of diatom material on the cover slip and let it dry there. Then I add a drop of Pleurax and heat to about 100, then 160°C. Then I pick up the cover slip with a warm slide and the warm fluid mountant spreads and fills the gap. I then let the slide cool with the cover slip hanging on the downside. This way i make sure that my objects are close to the cover slip and don't have to worry much about mountant volume.