This post is titled in honor of Stormy Daniels, who stars tonight on 60 Minutes. I will write here about “adult” rays, but the post will be totally safe for work. Indeed, it might help you have a nap…
Last week, I ran the second 300 nM oryzalin time course. It went well, insofar as everyone germinated on cue and nothing capsized or broke. At the end of the experiment, the roots looked perfectly plump and nicely similar to each other. Discovering the hidden disaster will await my measuring lengths and widths.
I also spent time recalculating data for a collaboration I am doing with a colleague in Nottingham UK. A few weeks ago, I discovered that I made a mistake (forgetting to convert the units appropriately). Unfortunately, this mistake is deep in the meninges of the dataset so I have to carefully undo, step by step. Lots of cutting and pasting in the spread sheet program. I made progress, getting about half way through all of the corrections. I will write about here when it is corrected all the way through.
But I do have images from my trip down to Brookhaven National Laboratory when I used the synchrotron. On Friday the 16th, I loaded the car with a shoebox-size box full of bits and pieces of lab gear, a travel-safe can of a special organic solvent, and a plate of Arabidopsis thaliana (“lab weed”) seedlings, 5 days old. After a little less than two hours to Bridgeport Connecticut, I got on the ferry to Long Island. Sunny and bright aboard, but too windy for the deck, I reached the Brookhaven Lab around 3:30 pm. I stopped in at the administration building, got my dosimeter, and then pulled up beside one of the many office buildings nestled around the circumference of the synchrotron.
Inside, I met up with Lin Yang, the beam-line scientist with whom I work. There are about 20 beam-lines around the main beam ring, each one emerging at a tangent. The beam-lines are designed for a specific type of investigation. Lin runs beam-line LIX16, which is dedicated to X-ray scattering for biological samples. The beam-line terminates in a hutch, a room-sized structure containing the sample holder and detectors. When the shutter is to be opened, bringing in the beam, the white metal doors of the hutch slide closed with fanfare (well, a trumpet call would be nice, they use a loud digital chirp instead). Beside the hutch is a row of desks with computers to run the setup and a bench at the end for preparations.
Our idea was to set up the gear Friday afternoon and then work on Saturday. First I had to put the plants in the small wet-lab room associated with LIX16 (and perhaps one or two others devoted to biology). The lab has lights just above the bench that are allowed to stay on overnight, so the plants can be photosynthesizing while they await their time in the beam. Now of course, to even set foot in the lab, I needed safety training. Mine had expired. A safety officer showed me where the phones and fire extinguishers are, and went through all of the various rules. This person was just as bored as me and we had a good time rolling our eyes over the earnest admonitions.
With the plants settled in for the night, I returned to the LIX-16 beam to set up my gear. I took a picture:
A crowded bench with the detritus of a 1000 experiments and repairs. The metal pipe at head level sweeping across the photo contains the beam-line. The pipe is smack in the way when you need to get the other side of the bench; you have to duck. Given care and attention the safety officer had just lavished on, for example, the precise disposal of sharps, this seemed a little … careless.
I brought a fair amount of gear with me from Amherst because what I wanted to do was tricky. I was hoping to see scattering from cellulose in root that remained wet. Cellulose being periodic and reasonably well aligned scatters X-rays, an interaction that has been exploited for years to understand the structure of wood. In wood, cellulose is far more plentiful and regular than it is in a growing organ, like a root. Still, a few years, a group published an analysis of cellulose orientation in the lab weed hypocotyl, while it was hydrated. With a bright and uniform source, i.e., the synchrotron, detecting scattering from a single hypocotyl or root is feasible. Because I study roots, I wanted to see if I could get signals from that organ. All of my work to date on the orientation of cellulose has involved cutting sections; the idea of getting information, even incomplete information, from an intact organ is tantalizing.
I wanted the root to stay wet because when it dries, the tissue wrinkles and this could well distort the orientation of the cellulose and also introduce scattering structures, such as surfaces brought into contact by a pair of wrinkles. But roots dry really quickly, especially the root of lab weed, which is little more than a tenth of a millimeter in diameter. A wee thread of a thing, 30 sec in air is enough to dry it out. It takes way longer than to put the sample in the beam and detect the scattering from a set of positions. Therefore we had to figure out how to keep the root wet. I had hoaked up a contraption involving thin wire loops and films of agar (wet) and a hydrocarbon called Formvar (evaporation barrier). While Lin watched, I cast some rectangles of Formvar on water, setting them aside until the next day for encasing a root on a wet film of agar. Everything was ready.
The next morning, Lin and I started discussing the ‘wet’ problem. He was fairly sure that agar would scatter X-rays too much, and I was worried that water would evaporate thru the Formvar. He came up with an alternative. He found a stiff sheet of plastic, I think it was a part of some clamshell packaging (the kind you can never open). In a small piece (about 3/4 inch by 3/4 inch) he punched a hole (4 mm or so in diameter). Then, he took a thin sliver of mica and glued that over the hole using an adhesive that sets up under ultraviolet light in about 30 sec. That formed a small chamber. I put water in this chamber, followed by a root, cut off the excess root, and then put a fresh piece of mica over the top. By doing this slowly and carefully, there were no air bubbles in the chamber and surface tension was good enough to keep that top mica attached.
Using this chamber we got scattering patterns from a wet root (Figure 2, top). Each little ‘bulls eye’ is a pattern that represents probing the root with a beam that is around 10 µm in diameter. The sample is then moved by 10 µm and a new pattern obtained. We did a line scan across the root, from clear water on one side to clear water on the other. To save space, I reproduce here only the part of the scan from the middle of the root. The key point is that the bulls eyes are circular. If the detector was picking up scattering from cellulose, the scattering patterns should be asymmetric because the cellulose is oriented more often in one direction than in another.
We took several scans on a few wet roots. To no avail—all of the scattering patterns were round. No hint of an asymmetric signal, as expected for cellulose. As a comparison, I dried a root down on the mica. Scattering patterns from the dried root were strongly asymmetric (Fig. 2 bottom).
This is disappointing because working with wet roots minimizes any distortions due to drying. And approaches that use sectioning (polarized light or scanning electron microscopy) suffer from the same problem. Still the strong signal from the dried root is a consolation. We decided to explore that further. The next step will be for me to dry roots more carefully, using the same kind of prep that I make for scanning electron microscopy. This is hardly as good as wet, but it will be many times better than rapid air drying. We can compare the X-ray results from carefully dried roots with what we see with polarized light microscopy, which I can readily do both wet and dry.