Yesterday I went to the biophysics lab, both to attend their weekly group meeting (another very good practice talk by a grad student) and to finally test whether I can see the cells under their microscopes (yes I could).
Their ordinary scope is a Zeiss that has darkfield but not phase contrast. We couldn't get the darkfield set up properly. Having now read up a bit on how to set up darkfield, I realize this was probably because we didn't have the 'stop' disk for the 20X objective, and didn't have a special darkfield condenser for the 100X objective. But the RA showed me how to get OK contrast by shutting down the condenser diaphragm to a pinhole, and with this I was able to see not only B. subtilis cells but the much smaller H. influenzae cells. This let me check that the cells were attached to the coverslip of the chamber before I put the slide with the chamber into the tweezers apparatus.
The laser component of the apparatus hadn't been realigned yet, but that didn't matter as I just wanted to use the visible-light component to find out whether I could see the cells. If I hadn't been able to see the cells I might have had to abandon the whole project. I guess I wasn't proceeding entirely out of optimistic ignorance, because I did know that the biophysics grad student who started this project a few years ago had been able to see H. influenzae cells in the apparatus he was using. I couldn't get the cells into very clear focus under the 70X water-immersion lens of the tweezers apparatus, I think because the optics were a bit out of alignment. The cells also weren't very conspicuous because they aren't very refractile (unlike the polystyrene beads). But I could easily see that they were there.
From now on I'm going to mark the center of the coverslip with an X using a fine-point Sharpie before I assemble each chamber. This X will give me a easy target to focus on, right in the plane of the attached cells. I did this today, with some more careful tests of cell attachment, and it worked very well.
Tomorrow I'm going to make more competent B. subtilis cells, and continue testing cell attachment issues, especially how to prevent the beads from sticking to the cover slip.
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