Welcome to the new look Lab Fab. Not my idea. Campus IT overlords decreed that Origin, the theme used by my blog, shall be discontinued. In blog-land, the “theme” means the set of design and layout elements that control what the blog looks like. Apparently, Origin is old (and hence insecure vis a vis hacking) and not popular (hence not worth the effort to anti-hack). I had to chose another. I did—you are looking at it—but only with the unstinting help of Graham Gilbert, a support specialist at Campus Press, the outfit UMass contracts to keep its blog platforms free of rust. As of the moment, the Lab Fab home page does not include a thumbnail from each post but Graham tells me that can happen and will happen. Please, do let me know if you have thoughts about the new theme.
No post last Sunday because I spent the preceding days writing a grant proposal. The opportunity plummeted on me like a hail storm in July: a collaboration with a scientist in India, Dr Jitendra Thakur. NSF put out a joint call with an Indian agency and he wanted to go for it. The idea is something I have wanted to do for more than a decade; so, despite the last minute rush and general feelings of “I don’t have time for this and they will never fund us”, I got out the quill pen, ink well, and penknife. Kind of bracing to have a writing immersion.
This past week, even with the proposal more or less dusted and done I could not go scuba diving thru the lab. I had to face duties I had ignored during the proposal lather; anyway, I could not do much with my twisty roots because the function on the confocal I need to tune up the calibration was still off line (see this post).
I did image some roots. I compared roots stained with fast scarlet and congored. I had never compared them side-by-side on the confocal, including checking the polarized fluorescence. Congored is brighter, need less gain (or laser power). But some of the cells stained by this dye (and none by fast scarlet) seem to stain oddly. I don’t know whether this is because individual roots vary or because of an issue with the dye. However, the main conclusion is that, again, I could see fibers and the apparent orientation calculated by the instrument matched my eyeball’s impression of which way the fibers go, closely but not exactly. This is what happened last time. This supports the cautious optimism of that previous post although I do need image the same root at multiple orientations to determine whether the discrepancy is a function of the root’s orientation.
And…the histogram function on the confocal got fixed. Thank you Alex! This turned out to be a dog’s breakfast, I think lunch and dinner too. The fault was deep in the meninges of the software. With that cured, I got in a new calibration run (Fig. 1). As previously, the y-axis is the angle calculated by the system and the x-axis is the known angle. Note that except for those at 45º, each symbol in the graph is in fact two symbols; I measured from 0 to 180º and back to 0º, in 15º steps. The replicate measurements are almost exactly superimposable. Points around 70º and 160º are a little bit off the line.. Note that again I have yet to implement background correction, which might inch values closer to the line. Working on that. I suspect this calibration is about as good as it gets.
Figure 1. Calibration test. Plot of the calculated angle vs the known angle for a set of images taken of the rotatable polarizer. Mostly looks good except for baffling weirdness at 90º.
With one screaming exception: 90º. Both points are miles off the line. One of the points at 45º is also far from home but since the other point at 45º is ok, I can write that off as some kind of glitch. But with two points at 90º, measured independently, being so wrong, I am baffled.
I am so baffled that I am going to forego theorizing. I measured these images only this morning. Maybe an apple will hit me on the head? If so, I’ll recount my concussion next time!