First objective test...or why did I get up today?

Klaus Kemp's 8 Form Test diatom set had arrived, the latest version of Canon's EOS Utility program was installed on my laptop, a green filter languished in my filter tray and I was ready to be amazed by views of resolved punctae of the diatom species Pleurosigma angulatum. I had just read David Walker's excellent article on his experiences with the diatom test set using, as luck would have it, similar objectives to those in my possession.

http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artsep08/dw-pa-test.html


All that was left was to do the deed.

Pleurosigma angulatum using a Zeiss Neofluar 25/0.60 with brightfield lighting and green filter

Pleurosigma angulatum using a Zeiss Neofluar PH2 40/0.75 with brightfield lighting and green filter

As shown in both photos, the punctae were not resolved, at least in the photos presented here. The second photo is clearly better but just doesn't resolve the detail that should be evident unless a lot of imagination is employed. So what went wrong? I'm not really sure but on reflection a number of things may have been responsible.

1. Focus. I had great difficulty trying to focus the specimen, even with the maximum magnification available via the EOS Utility image on my laptop. While I could see the image, and the detailed punctae through the microscope ( at least with the 0.75 NA objective) I could never focus that well on the computer screen.

2. Camera shake. The top shot was taken at 1/100 and the bottom at 1/250 sec. A Canon 40D was used in live view and silent mode was enabled. The shutter was activated via the EOS Utility software. Additionally, the body of this microscope could be safely used as a boat anchor for a larger boat than I could afford and that mass can absorb a lot of vibration. Although this issue has not been totally exonerated as the culprit, it has gone to the bottom of the list. Then again, there is the issue of the table the microscope is resting on. So camera shake may again be a serious contender.

3. Inadequate condenser. My condenser is an apl 0.63 NA version and would theoretically only affect the performance of the 40/0.75 lens. In further tests, I'll try condensers with higher NAs. I have two on my bench right now, a 0.90 NA and a 1.4 NA.

4. Poor objectives. Looking at both objectives through the optovar's Bertrand lens I've seen evidence of scratches and other possible issues. I have no idea how this might affect the resolution of my image but it has to remain a strong suspect.

5. Other causes. The objective image goes through more "glass" in it's eventual trip to the sensor and any of that could reduce resolution. Additionally, the camera is a complicated beast  and I may have inadvertantly screwed up some settings. I just reread David's article and another culprit, at least in the second, better image, might be a lack of contrast which might be provided by oblique illumination.

Can't really think of much else for now. Please remember that I'm a relative rookie at this stuff and the whole thing may just be a matter of technique. And if you have any suggestions, please don't be shy...share them.