We live in polarized times: the word polarization is starting to stand in for all that ails us. But today I can write about polarization that is happy, or at least useful (Fig. 1).
Figure 1. Images taken through a polarized-light microscope of a strand of xylem isolated from a celery stalk. Left panel: Strand is seen between two crossed polarizers (black background). Because the material of the sample interacts with the state of polarization (technically, the sample is birefringent), light is now able to pass through the second polarizer, so the sample is brigher than background. Middle panel: a compensator (first order red plate) is introduced with its slow axis as indicated by the cross at the top of the panel). Right panel: the compenstor rotated by 90º. When the sample is blue, its slow axis and that of the compensator are aligned. The slow axis is direction in which the molecules of the sample are aligned and most strongly interact with light.
One day the week before last, I left the lab, walked along the towpath beside the canal until reaching a Sainsbury’s supermarket. I bought celery. Back at the lab, I nested the package in two plastic bags to avoid scenting the air, labelled the outer bag, in bold, “Do not eat! For experiments” and stashed it in the cold room. This past week, I retrieved the celery from the cold room—no one had nibbled—and prepared xylem.
If you have munched celery, you will be aware that the stalks are stringy. In fact, recipes call for de-stringing the stalks to promote smoothness. In fact, the strings are vascular bundles. I removed a stalk, cut off each end; then, with the two curved edges of the stalk facing down, I snapped the stalk upward, cracking it through, except for a thin layer facing me. I could call this the back of the stalk; a botanist would call it abaxial. Carefully, I peeled the layer along the length of the stalk to the cut end. This layer is a sheet of tough epidermis and adjacent tissue, embedded in which are those those strings. They are easy to see (Fig. 2).
Figure 2. Strings in celery. Left panel: strings being lifted out of from the outer part of a celery stalk. Not my picture, rather from here. Right panel: a few strings after the treatements described here. Again, not my picture. This one comes from the paper where I got the protocol: Gray Cellulose (2014) 21: 3181–3191.
Next, using a fine tweezers (and sometimes my not-so-fine fingers), I pulled each string out from the enclosing layer. The string holds together, allowing it to be pulled out of the surrounding tissue, because it is tough. The xylem conducts water under considerable tension, a job that requires toughness. There are likely to be many plants with long tracks of stringy xylem that could be similarly pulled out; maybe not so many for sale at Sainsbury’s.
I collected a few dozen strings in a beaker of water. I boiled them for 30 minutes. Boiling happened over a Bunsen burner (I prudently brought in matches to light the thing—I hate the flint-strikers which spark impotently in my hands); the operation smelled like soup. Had I been hungry…I suppose. The strings held together fine. Then I soaked them in 5% sodium hydroxide for an hour. The boiling water and the strong base removed material from the previously living cells surrounding the xylem. The tough water-conducting xylem cells are dead in the plant. After rinsing the strings with distilled water about eight times, I soaked them in ~10% sodium chlorite (no, not salt, but NaClO2 – a bleaching agent) for an hour at 37ºC. I did this in a fume hood because in fact the protocol required me to add acetic acid to the chlorite and this is said to give off chlorine gas, a scent well worth the trouble of avoiding.
After that, again, I needed too many rinses in water to count. I stained some of the now white strings (Fig. 2) in fast scarlet and others in calcofluor and mounted them for microscopy. Mounting required using fine tweezers to break up the strings into something closer to single strands of xylem (Fig. 1). To take a quick look, I got access to a polarized-light microscope in the campus Dry Lab used to teach geology. Rocks comprise minerals with microscopic crystal domains and so in Geology, polarized-light microscopy is common.
The xylem look great (Fig. 1). The magenta color is produced by adding a compensator to the light path; this has the effect of removing green light from the incoming white. The remaining light is blue and red: the magenta in the background. In the xylem strand, the color changes from blue or yellow when the compensator is rotated by 90 degrees. The color changes because in one orientation, the crystalline material in the sample reinforces (that is, adds to) the effect of the compensator and in the other direction the sample opposes (that is, subtracts from) the effect of the compensator. When the compensator is reinforced, red is removed from the incoming light, making blue; by contrast, when the compensator is opposed, blue is removed, making yellow.
I prepared celery xylem not because I wanted to stare at the lovely magenta (although I admit to some staring). I need a sample for equipping a confocal fluorescence microscope with polarized light. This will require a fair amount of what the Brits call faff. To see whether the system runs right, I need a sample with a strong signal and one where I know what to expect. Xylem is bright in polarized light and well characterized. It fits the bill. Now, on to the confocal…