Sept 18th: Mounting tension

With a set of new sections from Joe Hill rotating slowly on bench, and no obstacles looming, it was time to see what a fresh set of samples would do. On Monday, I moved them from ethanol to t-butanol. The sections sit in small glass vials, submerged in a few hundred micro-liters of liquid. Holding the vial at an angle, I slip in a long, thin glass pipette (which are named after Pasteur) and withdraw as much of the solution as I can without at the same time sucking up any of the sections. Then using the clean pipette I add fresh liquid. This is fairly easy, although I do have to fossick around to find my glasses to give me a fair shot at seeing the whiskery sections.

The sections soak in t-butanol all day Monday, with four fresh changes to make sure that all the residual ethanol is long gone, and then overnight. On Tuesday morning, after one more fresh change, I put the vials in the fridge, where the t-butanol freezes, turning a waxy white. After a few hours, I take the vials out of the fridge and put them under the bell jar of a vacuum pump and let it rip. Under a vacuum, atoms at the surface of the solid t-butanol turn to gas, without passing through the liquid state. This is sublime. No really, scientists term the direct passage from solid to gas sublimation. The sublime treatment avoids exposing sections to a liquid surface and to the concomitant surface tension forces, which are mighty and destructive. After about an hour, all the visible t-butanol has gone, leaving the sections exposed and dry at the bottom of the vial. I let the vacuum run for about thirty minutes more to ensure that all the t-butanol is pumped out.

Dry samples are essential for the SEM but they cannot be imaged in the bottom of a vial. The sections need to be placed on a metal stub. The stubs are a little more than a centimeter in diameter. I start by sticking on the stub a rectangle of double-sticky carbon tape. This tape is conductive, so that electrons can readily move from the section to the stub itself and onwards to ground, and sticky so the samples will be motionless. I make the tape rectangle asymmetric by removing one corner. Then, I place the sections on the stub. This is harder than it sounds because the sections come out of the drying vacuum with gentle bends. They need to be flattened down. Also, the tape is super sticky so there are no second chances when a section hits in the wrong orientation or with the wrong side up. Finally, I draw a picture in my lab notebook for reference. Or sometimes, take a picture:

Five sections mounted on a stub for imaging in the scanning electron microscope. The circular shiny material is the top of the aluminum stub. The black rectangle with a corner missing in the lower right is the sticky carbon tape, on which the sections are stuck.

Five sections mounted on a stub for imaging in the scanning electron microscope. The circular shiny material is the top of the aluminum stub. The black rectangle with a corner missing in the lower right is the sticky carbon tape, on which the sections are stuck.

For this stub, made some time ago actually, the sections got fairly beat up. I am happy to say that Joe and I have figured out better methods for handling these thigns and the sections that I am working with now hold together pretty well. The center part of the sections contains pith. This is soft and fragile. The outer part has the fibers. This part is strong and generally intact. Fortunately, this project focuses on the fibers.

With the sections mounted, I booked time on the microscope. Ahhh—I could get on for just an hour on Friday, because the machine had been down during the first part of the week. But an hour was enough time to get some useful information. I found these sections imaged more reliably than ones I had been banging my head on during the summer. This was a big relief and I got useful images. However, this new set of sections still did not image as easily as other kinds of plant sections I have worked with over the years, even from the same material. It seems that there is something intrinsically difficult about working with the stuff sent up by Joe. He cuts them thicker than ones I have handled before, and cuts them on a cryostat, which requires infiltration with magic cryostat goo. Perhaps either of those things makes them difficult to coat with platinum properly? In addition, having them sit around the lab dry seems to make them even harder to image. Maybe they adsorb water? Not sure. What I will do is to mount a few more stubs and coat them now, on the theory that this might prevent or at least retard this unwanted aging phenomenon. I am hoping that by working assiduously this week and maybe next, I can finish the collection of images and can get on to analyzing them. What are they telling me about the cell wall??