May 14th Benched

Almost two months since the last post. I have been occupied with paper writing, teaching, committeering, traveling on some weekends, which is when these posts usually get written. I considered blogging about paper writing because, after all, LabFab is trying to convey what a scientist does day in and out. Paper writing is an essential part of the life scientific. But how do I describe a bunch of edits to a paper, without simply repeating them? I like writing about actions and apparatus, things with color, shape, texture, and weight. The actions give rise to thoughts, which balance and enliven each other. Where is the balance were it only thoughts? For now, I will continue to focus on the doing of things and with any luck weeks without lab work will be few.

Print by Hercules Segers. This has nothing to do with this post, except that one of the things I did over the past two months while NOT blogging was to see the show of this amazing artist’s prints at Met in NYC. Spellbinding.

Still, I did do a little bit of lab work over the past two months. I didn’t post about it because it was, in a word, boring. Perhaps writing about the synchrotron  raised my expectation bar? That is something I need to change – from now I will write about time in lab, whether the upshot was a sonic boom or a whimper.

The past week’s work started the week before but got going in earnest Sunday. The previous Wednesday I had set up some maize seeds in jelly rolls. That is, I laid out a narrow rectangle (say 2 inches by 10 inches) of spongy absorbent paper, wet it thoroughly, added a paper towel, put a row of ~20 maize kernels across the top (long) edge, added a second paper towel, and then rolled the thing up. Sorry I don’t have picture. This roll is then stood up inside a plastic box, with a centimeter or so of water in the box to keep the roll wet. The kernels are all at the top edge of the roll, meaning that the roots can grow straight down, between the paper towels. I used to germinate the seeds flat on the bottom of the box and then the roots curve all over the place, trying to grow down. I was in the lab on Sunday because the roots were mostly 4 to 7 cm long, about as long as they can get without reaching the bottom of the box. Ready for sectioning.

I had a fresh crop of maize roots is because for my next outing on the synchrotron I will X-ray sections. We will use dry sections because then we don’t have to worry about them drying out while we watch, as the X-ray signal is collected. Also, with things the size of these sections, the angle at which the beam hits them can be adjusted more freely than with a long thin root. Because drying biological things causes distortion (think cotton clothes coming out of the dryer, only way worse), I will dry the sections as if for SEM, which minimizes distortions. Hey – waitaminnit! Since I am drying for SEM, why not take a sample section and look at it under the SEM? That way, I can have a direct image of the net fiber orientation of the samples that will be X-rayed? Sounds like a plan.

I cut sections that are 100 µm thick. This is about the thickness of the entire arabidopsis root and it will be a good test of the sensitivity of X-ray beam. I cut them from two different places in the root: near the tip and about 5 cm away from the tip. The ones near the tip will have cellulose oriented mainly transverse to the long axis of the root and the ones farther away will have cellulose oriented in helices. That will give two distinct fiber deployments with which to challenge the X-ray system.

To cut sections, I first take a roughly 5 mm segment of the root and glue it onto the Vibratome stub. This is the only part of the business that makes me nervous because the glue dries fast and any false moves will cause it to dry down at the wrong angle. The sections are cut in saline (i.e., PBS) and then immediately incubated in saline plus detergent (i.e., Tween) to wash away leftover cytoplasm. I collect the sections in 6-well plates (each well holds about 6 mL of fluid). No reason to keep sections from different roots separate, so there were two wells, one with the tip-region sections and the other with the farther-back-region sections, about 15 of each.

The sections get dehydrated progressively to 100% ethanol and then the ethanol is exchanged with a different solvent, t-butanol. Then the samples are put in the fridge for 2 h, which solidifies the butanol. Finally, the solid butanol containing the samples is put under a vacuum, which causes the butanol to sublime, leaving behind the now perfectly dry sections. For dehydrating, I remove the solution in the well with a small plastic dropper, being careful not to remove any of the sections, and then add the next solution. I call this “drain and fill”. But after the last change in 100% ethanol the samples need to go into small vials and so I have to move them one by one with a pair of fine forceps. But after they are in the butanol, further changes go by drain-and-fill in the vials. The vials are placed in the drying apparatus directly, only the lids are replaced with holey lids so that the butanol can be pumped out without at the same time pumping out the sections. I learned to do this the hard way.

I dried sections on Wednesday and I will mount a pair of them for SEM this coming week. Also I had set up more maize seed on Saturday so that on this past Wednesday I cut more sections. This time, I cut them 400 µm thick, to have some fatter ones on hand in case the synchrotron has trouble with the thinner ones. These are sitting in ethanol right now and will be dried this week.