linuxusr wrote: ↑Sat Nov 13, 2021 10:50 pm
-- I tried measuring the depth of a slide, using top and bottom markings, without a coverglass. The slide was 1,130 μm. I had to rotate the FF knob so many times that it was hard to count, so I abandoned that and went back to the coverslip marked on top and bottom with a marker. There are tiny specs within the marker liquid from which you can get a sharp focus.
Also, I measured two marker strips with no marker strips and the caliper readout was the same. Probably that depth is in nm and can be ingnored.
From the top to the bottom of the coverglass I passed through the range 77-18 = 59. 150 μm for the depth of the coverglass /59 gives me 2.54 μm which is the distance between two integers on the FF knob on the Z axis. This is the same value I got the first time except for 4/100 or 1/25 of a μm which is negligible.
It seems that the marker method works well! It's probably easier to focus on the ink specs than on the edge of the glass. Did you divide the calculation by the refractive index of the glass? Considering that the refractive index is around 1.5, it would make a difference I think.
linuxusr wrote: ↑Sat Nov 13, 2021 10:50 pm
Unless you think this value is really, really suspect, I think that in the future, if I want to know the depth of a specimen, I'll focus on the top and bottom, take the difference between the two numbers and divide that into 150 and that should give me Z axis μm through the specimen.
To calculate an accurate specimen thickness, you have to multiply the change in stage position by the [refractive index of the specimen divided by the refractive index of the coverslip], as the bottom of the specimen is observed through the specimen and has undergone refraction by the specimen, while the top of the specimen is observed through the coverslip. According to my thin section microscopy guide, you should know the refractive index of the specimen to at least the first decimal.