Siliceous and Delicious

Spent the morning trying, at long last, an old idea on the CLSM front: making the epoxy fluoresce (rather than the fossils). It’s an exciting idea, because—it it were to work—it would actually yield images of the whole skeletal volume, rather than just an image of the surface covering the skeletal volume. However, as with everything I’ve tried, there appear to be challenges.

First, neither straight fluorescein nor the fluorescein sodium salt will dissolve directly in either of the epoxy components. So, I tried adding to the epoxy fluorescein sodium salt (which appears to be far more soluble in general) that had first been dissolved in a small volume of either ethanol or DI. The resulting epoxy is wonderfully fluorescent (when illuminated, it glows bright green like the radioactive brick in the Simpons titles sequence) but seems to form bubbles no matter what. I tried lowering the curing temperature from 80˚ to 60˚C, and this prevented bubble formation in a blank trial slide—but bubbles still formed when I attempted to prepare a slide with fossils. Under the microscope, it appeared that each individual bubble was nucleated on (or at least closely associated with) a fossil, making matters worse.

I had to stop after a couple of hours to turn my attention to an even more pressing task: writing the next EPS 8 lab. Jc and I sat down and together wrote the first part, on the Signor-Lipps effect, fairly quickly. The rest went substantially less quickly. We got only part of the way through the P/T extinction section before the day was over and it was time to head out for my squash game at 6 pm.

After squash I returned to work, having scheduled time at the CLSM to see if my inverse-staining method worked. It was a frustrating exercise—for the first part of the evening, I seemed to be able to get some images, though the matrix fluorescence was far from even throughout the whole background. Rather it seemed that there the fluorescence was bright in the center of the field of view and decayed to almost no fluorescence at the edges. The non-fluorescent areas at the center of the image—constituting the frustule structure I’m trying to image—were poorly defined and not nearly as crisp as the images I’ve been able to obtain by staining the frustule. What’s more, focusing to the bottom of the preparation—where the fossil sits on the coverslip—brings the plane of focus to a point where it appears not to intersect any epoxy, as the fluorescence disappeared altogether, leaving blackness and consequently no image. The most frustrating part ensued when I tried to rectify the situation by changing the filter set-up, after which the image disappeared altogether and all I seemed to be able to get was either a black screen (no light reaching the detectors) or a fully saturated screen (the laser directly hitting the detector). I wasn’t able to make the image reappear after that.

One final observation, and another strike against the inverse-imaging idea, was that the stained epoxy seemed to bleach quite rapidly. After just a couple of minutes of scanning a specimen (in the process of trying to get the z-stack set up right, which incidentally is also a total pain in the backside with the Olympus software), the fluorescence started to wane, and when I moved the stage by a small distance, the previous edge of the scanning field was visible as a bright field of fluorescence, showing starkly by contrast how much the scanned area had bleached.

In summary: as much as I’d fallen in love with the idea over the past few days, I’m no longer convinced the inverse-staining method is the solution to my problems. I will try one more time, with Evangelos’ help, and then decide.