LouiseScot wrote: ↑Sun Nov 15, 2020 2:07 pm
I'm afraid I'm not a very good judge of these things. I thing it's quite a difficult subject since the contrast is low. I've adjusted contrast and brightness somewhat. Also, Nikon 20x/0.75 is difficult to focus because it has a miniscule depth of focus. ... Someone mentioned previously I had a lot of spherical aberration but I'm not really sure how to tell?
Yeah I think it is hard to judge just looking at overall appearance of photos. Spherical aberration can explain a general hazy look and lack of contrast ("soft focus" in photography) but so can a lot of other things. Photographs of scenes with small, bright specular highlights can clearly demonstrate the effect on points shown in
https://en.wikipedia.org/wiki/File:Sphe ... n-disk.jpg but not so common to see small, bright highlights in bright field transmitted-light images.
The relatively poor contrast of the Olympus 40x compared to the swift was maybe suspicious, but with the other variables (different camera) hard to say.
May be easier, rather than trying to diagnose from the look of the photos, to experiment with some other common causes of problems. With positioning of tube lens ruled out, might be good to look into cover glass thickness next? I think you mentioned elsewhere this was an antique diatom slide? Have you tried to establish how close the depth of the diatoms below the cover glass surface is to 0.17 mm? May not explain the 40x Olympus/Swift difference, unless the Olympus is higher NA, but critical for your 20x 0.75. Have you seen figure 3 here:
https://www.microscopyu.com/microscopy- ... correction
If your motorized focus rail is calibrated (can be moved known, relative distances) you can measure the depth of the diatom by seeing how far you need to move it to go from focusing on defects/dirt on the surface to focusing on the diatom, then correct for plane-parallel plate effect of the cover glass and mountant on the focal distance. (At least, I think that works. I experimented measuring cover glass thickness that way on my Reichert Microstar IV and got reasonable results comparing against a micrometer.) If you try this, best to move the focus carefully in one direction only through both focus points to avoid inaccuracy due to backlash.