I am living the cliché: it comes together at the very end. I hope that I don’t come apart.
On Monday I caught up on a few things because the roots were too short. When the plate with seedlings is placed on the horizontal microscope, I can move the stage down only so far. With short roots, the stage doesn’t down far enough for the root tip to enter the field of view. Dang it! Of course, had I expected this, I could have placed seeds lower on the plate; that’s the clarity of hindsight. My new growth cabinet, or something, is not the same as before. Still, I didn’t mind; I had plenty to do.
On the remaining weekdays, I put a set of roots through the imaging workflow. Each day, the workflow handles three roots. All told, the workflow takes about 5 hours; a full day, intense but fine. I did wild type twice and tua4 twice. I also compared the two dyes: congo red and fast scarlet. The former needs to be used at a ridiculously high concentration but gives much brighter staining. I wanted to see if there is any difference in the anisotropy of the signal. No, not obviously; but the greater brightness tends to improve the overall quality. So, congo red wins. I will try to back off at least slightly on the concentration because there is brightness to spare.
Pushing roots thru the paces feels good. The problem is: I don’t have time to analyze. Well, I suppose I could analyze after dinner and over breakfast. Forgive me, but no, I can’t. I like having a life, and a wife; both need my participation. The analysis will simply have to follow, someday in the autumn. This makes me uneasy. What if none of it makes any sense? Or more realistically, analysis will inevitably reveal better ways to have collected data. In the ordinary way, I would be going back and forth between analysis and data acquisition, a dance that allows for step-by-step improvement. But not this time.
The way I have been seeing what is going on is thru a qualitative check on the polarization. The confocal saves the input images in a proprietary format. For the analysis of polarized fluorescence, I have to convert the set of five input images into another format for the software that analyses the polarization. I add a conversion session to the workflow, after I have collected the confocal images. Conversion is as boring as waiting for a bus—click, click, and click again—but I do look quickly at the results for each image set (Fig. 1).
Figure 1. Example of polarized fluorescence from a root. Images show a region (~100 µm by 100 µm) of the root where root hairs are beginning to emerge (the bright bulges at the ends of some cells). Upper left image: An input image: fluorescence is from congo-red stained cellulose, the exciting beam is polarized horizontally. Note the faintly fibrous texture of the cell wall in a few places. Upper right image: Same image as upper left but with lines superimposed showing the calculated average orientation of the dye molecules. The cellulose is running obliquely or longitudinally in most of the cell walls. Lower left image: Calculated anisotropy of the dye molecules: the brighter the signal, the more aligned (i.e., the more anisotropic) are the molecules. Anisotropy is high along the side walls because, unless the cellulose is perfectly perpendicular to the page, there is always a component parallel to the cell. On the cell faces, anisotropy is low but not zero. Lower right image: Orientation lines superimposed on the anisotropy image.
Clearly, extracting quantitative data from these images mapping the orientation of cellulose vs position in the root will be … fun. Some cells seem well imaged but others are a little out of focus, being a little deeper than, or raised above, the others. I am mulling over a criterion for dividing cells (or regions) into measurable vs not. For example, note in the background around the root, beyond the nicely imaged cells, the anisotropy is salt and pepper, indicating noise; whereas, the orientation lines are uniform. Perhaps I can use anisotropy values to constrain the regions available for angle measurement?
Maybe. But for now, it is just a question of how many roots can I stash away on the hard drive. For the coming week, on three of the days for imaging, I am going to double dip, staring around 9 AM and finishing after 7 PM. Two imaging runs in the same day. Six roots!! I am not sure I can handle this but sample size matters—I’ll try. Go big or go home? Or in this case, go big before going home!