Time to mind the whys and wherefores

            Last time, I regaled you with a story of cutting sections of roots embedded in paraffin. Cutting sections is the kind of obsessive handiwork that made me happy to become a scientist and still makes me happy to be one. I enjoyed getting roots into molten wax and I enjoyed blogging about doing that and cutting sections, so much so that I said nothing about why I was sectioning. I suppose, just to have fun with wax is not reason enough. Here then follows a brief account of what I am trying to do.

The panorama: I want to study the organization of cellulose in the primary cell wall by using X-ray diffraction

Starting at the end of that sentence, X-ray diffraction is a powerful tool for studying how the atoms pack together into structures. For some years, I have been using polarized-light microscopy and scanning electron microscopy, methods that examine structure at larger scales than the atomic. Nice, but now time to move down a level: X-rays, yay-rays! 

Polarized-light images of a section through the root of lab weed (Arabidopsis thaliana). The left image shows how crystalline the cellulose is, the more crystalline the brigher the pixel; the right images shows the orientation of the cellulose coded by angle. The inset radial structure helps you see how a given color relates to a given angle (over a 180º range).

Next back is the primary cell wall, which you might think means the best one. I do, but that is not how the term is defined properly. Instead, primary here means first; the wall that is built while a cell grows. After that comes the secondary cell wall, which is built in some but not all cells after growth stops. The secondary cell wall is made after growth stops because it is massive, hundreds of times, maybe thousands of times, more massive than the primary cell wall, certainly unable to be stretched by growth. Everyone is more or less familiar with secondary cell walls because they constitute wood, along with some air spaces. Naturally, the structure of cellulose in the secondary cell wall has been studied 960 ways to Sunday; the primary cell wall, not so much. 

I am interested in learning how the cellulose in the cell wall influences the growth of a cell and having looked at the cellulose with polarized-light microscopy and scanning electron microscopy, it makes sense to try X-rays. For some time, I have been trying this with primary cell walls prepared from tobacco BY-2 cells. I have posted about it, here and here. BY-2 cells are a population of growing cells, without distinct tissues, making them a good material. I have been removing the cytoplasm from the cell wall, removing most of the stuff besides cellulose from the cell wall, and sending the cellulose-rich remnant to Brookhaven National Laboratory for X-ray analysis. 

The bog-standard way to do this is to make a big mat of tens of thousands of cell walls and send the X-ray beam through. We have using such preparations and the results are fine. But along the way, my collaborator at Brookhaven, Dr Lin Yang, has spurred me to try examining single cells. Sensible because with one cell, we can study the relationship between the organization of the cellulose and the geometry of the cell; but challenging, because there is far less than a salt grain’s worth of cellulose in the cell wall of a single cell. But Lin works at Brookhaven’s Synchrotron, which has a science-fiction level bright X-ray beam so we tried. And amazingly, we got diffraction patterns from cell walls from a single cell. Cool.

But, and there is always a but: X-ray analysis works best on dry samples, because water scatters X-rays too well. To send an X-ray beam through a dry cell wall from a single cell, I took isolated cell walls and deposited them on an X-ray lucent substrate, a thin wafer of silicon-nitride. In life, BY-2 cells have the shape of a thin and twisty, roughly cylindrical pressurized balloon; when the cell deflates in death, and the cell wall is dried down on a flat surface, the wall wrinkles up, the excess surface area has to go somewhere. Also the top wall and the bottom wall collapse on top of each other, there being no way to rip open the cell. So, while the X-rays detected cellulose, the organization was marred by all the wrinkles. 

Finally, we get to the roots and the wax. I want to put a section of a root in the X-ray beam. By embedding roots in paraffin, I can cut sections (~8 µm in thickness) that are thinner than the typical diameter of the cells (in a maize root, around ~20 µm). When I look at the section in face view, many cells look like windows, with the cell walls on top and bottom of the cell being missing (that is, the 8 µm slab of section contains a central part of the cell); but here and there, the section contains the cell wall between the cell and the one below (cases where the section slab contains the side-edges of a cell). You can see both kinds of cells in the polarized-light images above. Cell walls captured within the section are ideal for an X-ray examination. The intact section will keep the wall from wrinkling up (too much). And the geometry of the section will make clear the relation between the cellulose orientation and how the root was growing before it was encased in wax. 

So. I am sectioning maize roots to expose the glory of primary cell wall cellulose for X-rays. And if you remember from last time, cutting the sections turned out to be a walk in the, if not park, at least parking lot. But, there is that but again, the difficulty comes after the sections are cut. And quite a difficulty indeed. Not to get all click-baity, but I am going to hold off explaining until the next post, when I will recount how I am  trying to squeeze some ade from that particular lemon. 

Still Life with Lemon and Cut Glass, Maria Margaretha van Os, 1823 – 1826. Rijksmuseum