I missed last Sunday’s post. All day Saturday was the Plant Biology Graduate Program Symposium (check it out here) and on Sunday from 10 to 3 was North Star Learning Center’s annual retreat for the Board of Trustees. North Star is worth a blog post all to itself but in the meantime read about them here. With the remains of the afternoon needed for shopping, last weekend provided no luxurious mental space for the weekly recap. In contrast, today is in the middle of three-day weekend, it is raining, and no social events. Perfect blog weather.

Over the past two weeks, I continued to work on the cell wall project for Joe Hill but I started analyzing images. This quickens the pulse. I have been collecting images since early spring, and despite the various difficulties, have amassed a goodly number. I decided it was time to see what it all meant.

The first step was to organize the collection. I can remember as a child my dad giving me envelopes full of postage stamps, which I then put into a stamp album in the correct spots. This is straightforward because the album provides the organization (British Rhodesia, three-quarter profile of King George V, 1 shilling, Nile blue, perforations 14 to the inch). For the cell wall project, I had bulging folders of images, but no album. The first step was to determine an organization.

One level of organization was extant, namely the imaging date. This is important because it corresponds to whatever is written down about conditions and the like in my notebook. But happily because this is such an important datum, I include the date of imaging in every file name. But the folder for any given day contained images of various qualities, magnifications, and treatments. The first thing I did was to separate them by treatment, which in this case means genotype. There are five of those: the wild type, three single mutants, and a triple mutant. For each genotype, the heart of the collection are images taken at 65,000 times magnification, one per cell. Those are special because it is from them that I hope to assess what the mutants are doing to the structure of the cell wall. Therefore, I divided the images into those taken at 65k and all the others. The others get saved for later.

Next, I looked through all of the 65k images and separated out the ones with charging artifacts. The word charging you might recall from a previous blog post is the scanning electron microscopist’s term for bad. For each genotype, this left me with about 40 good 65k images. The next thing to do was to run the meta-data extraction code that Mike Pound came up with (described here). This worked (!) and then I needed to see whether the images I had dubbed 65k in their file names actually were taken at 65k. Finally, I divided the good and true 65k images by the section they came from. That way, I could compare all the sections from plants of the same genotype.

Starting with the wild type, I immediately learned something valuable. The apparent variation between sections was greater, much greater, than between the cells of one section. To put this another way, if I showed you all forty sections and asked you to put them into four groups based on similar appearance, you would have a good chance of grouping them by section correctly. I also decided that there was a reasonable biological reason for this.

This figure shows a cross section through the kind of stems I am working with (inflorescence stems of arabidopsis). The cells with the thick blue cell walls are the cells of interest. The outside surface of the stem shows up in the lower right corner of the right-hand image. It has a thick cell wall too. Interior of this cell wall are three (or so) tiers of thin-walled cells that contain numerous small round spots. These are chloroplasts: the stems are green and photosynthetic. The stretch of epidermis in the right-hand image even includes a stoma and a sub-stomatal cavity, which help bring in the gas for carbon fixation. Interior to this are a band of thick walled cells. These make a ring several cells deep that encircles the stem. Their thick cell walls provide reinforcement that helps the stem stand up against gravity and wind. Oddly, the ring deviates around a bolus of thin-walled cells. The bolus is NOT a tumor, it is the phloem, the living tissue that carries sugar and other nutrients around the plant. And in fact, the thick walled cells immediately adjacent to the phloem are xylem, the water conducting tissue. Xylem and phloem together are called a vascular bundle. Summing all this up, moving around the circumference, there are thick walled cells in the xylem and thick walled cells, between xylem. The latter are termed fibers, and are dedicated to mechanical support.

I have been imaging longitudinal sections (i.e., cut lengthwise through the stem, at right angles to the images shown above). Because the sections are straight, they follow a given set of fibers. I already know that the thick walled xylem cells look entirely distinct at 65k. I can tell they are xylem easily from the special perforations in their walls to facilitate water movement. What I suspect is that fiber cells closer or farther from the xylem differ in cell wall structure; therefore, a given section represents essentially two position around the ring (one on each edge of the section). Similar ring-position translates to similar cell appearance.

Whether or not this explanation is right, the fact that I can see a clear pattern of more variation among sections then cells means that my collection needs images from a reasonable number of sections.

Alas, I didn’t have them. Two of the genotypes had 5 sections with at least 7 or 8 cells each, but I was short on the other three. I had actually imaged that many sections and more, but their folders contained only a few 65 k images. The variation among cells is hardly zero so it is essential that each section is represented by enough cells.

All of this spelled more imaging. I mounted up some new sections to fill the gaps, coated then with Xena, who by the way was beneficent and ran beautifully, and had two hours imaging. I filled collection gaps, with more to be captured this week with luck.

Now, besides all of this happy stamp collecting, I also started analyzing the wild-type images quantitatively. Numbers! In fact I spent the better part of a day doing this. But given this post is already soporific, I shall hold the numerology until next time.