Please enjoy a slice of pineapple upside-down cake on my behalf (Fig. 1). I solved my calibration issues by inserting the liquid crystal slider upside down.
Figure 1. Photo of a of a pineapple-upside cake, as shown on a BBC’s food page. Looks right side up to me; maybe there is a lesson there?
For months, I have been washing up against the rocks of calibration. I need to calibrate the liquid crystal unit before I can measure anything on the confocal fluorescence microscope. Calibration failures have dominated Lab Fab posts (e.g., here, here, and here). I worried that the unit was broken (one test I ran pictured here). I started to adapt the tech for a multiphoton microscope but misgivings about calibration remained.
Calibration on the confocal involves passing the light from the laser through the liquid crystals, then through a rotatable polarizer, then through the microscope’s condenser, and finally to a detector. You can think of the detector as simply a camera because it forms an image of the rotatable polarizer; but remember, because this is a laser scanning confocal, the laser is scanned across the sample (in this case the rotatable polarizer) point by point and the detector receives light from each point in turn and builds the image.
Last month, my confocal calibration saga was interrupted because the transmitted light detector failed. Even worse, the confocal in question, the Zeiss LSM780 in the imaging suite of the University of Birmingham’s Medical School and run by the redoubtable Alex Di Maio, is on the “do not resuscitate” list; in relative terms, it is older than me. Fortunately in Bioscience where I work, there is a Zeiss LSM710. When I was first organizing matters for my project, the imaging facility in Bioscience was unstaffed. I worried about the performance of an orphan ‘scope. However, now the facility has a manager, Tzer Lim. After introductions, I installed the software the software on the 710 and rolled up my sleeves.
The results were worse than on the 780. I became ever more certain that my project was doomed. However, in between previous calibration sessions on the 780 and now, Rudolf Oldenboug and Amit sent me a slide that they made for calibration. This slide has four pieces of thin polarizing film, arranged with the four axes of transmission at 45º. That is, one film goes at 0º, one at 90º, one at 45º, and one at 135º; they meet in the middle (Fig. 2). This slide is beautiful because when the liquid crystals are adjusted to give light polarized at, for example, 0º, the film oriented at 90º will be dark, the one at 0º light and, crucially, the ones at 45 and 135º the same intermediate shade.
Figure 2. The slide with four pieces of polarizing film. The orientation of the films, clockwise from the lower left are: — , / , \ , | (those front and back slash should be 45º and 135º). Image is taken with the incoming light polarized horizontally (—), so the lower right film (|) is dark. This image is taken with successful calibration.
At first, when I imaged this slide after calibrating, the settings for 0º and 90º seemed more or less ok but the ones for 45 and 135º were hopeless. It was almost as if there was hardly any action from the liquid crystals at all. Rudolf realized that these results looked like the crystal unit was inserted the wrong way up. Crucially, the quad-slide made this clear.
Rudolf is brilliant and his assessment followed the logic. But in terms of practice, it was crazy. For one thing, I inserted the slider here in the same way I had inserted it into the LSM780 confocal at Marine Biological Laboratory (Woods Hole), when I worked there, upstairs from Rudolf’s laboratory. And, it is the same slider: I borrowed it for the year. For another thing, although the slider, when turned upside down, does fit into the slot on the microscope, it is obviously “upside down” insofar as the slider housing is unfinished on that side and the liquid crystal unit itself falls out of the housing. Clearly, the thing was designed to be inserted right side up.
Nevertheless, with care to keep the liquid crystal unit in place, I inserted the unit upside down. Calibration worked (Fig. 3). I did not have time to conduct a full on xylem rotation series (there was a software glitch that took Amit about an hour to track down to a missing backslash) but a few trial images suggested that things are reasonable (Fig. 4). I can scarcely believe it.
Figure 3. Image calculated by the software with the orientations of the four polarizing films indicated by red lines. Note that the direction of the lines follows that expected from the known transmission directions of the polarizing films (see caption for Fig. 2).
Well, as much as I would prefer to use the multiphoton for the sake of the biology (roots inside the plate, staining with Calcofluor), getting the multiphoton validated seems likely to take about as much time as I have left here, with no guarantee of success. I will keep up with that. But for the main, full speed ahead with the confocal. There is just about enough time, provided of course, the rest of the universe cooperates.
Figure 4. Image of celery xylem stained with fast scarlet. Image shows the average fluorescence with the calculated net orientation of the fast-scarlet molecules shown as red lines. Note how the red lines follow the orientation of the ribs. There is a bright ‘haystack’ shaped blob under the xylem in the upper left that is probably scrunched up parenchyma.
To think that was the problem all along… congrats, Tobias! I’m stoked to see what you come up with.
p.s. the cake is baked upside down. Not sure what that means for the lesson of this chapter!
Well–I guess its ok as long as the cake is not half baked!
Strange that flipping it over would make a difference, not sure how that crystal does it’s job but super glad that you figured it out. Fun to hear about your progress, hope you can get done what you want to before your sabbatical is done.
Thank you Sarah! The unit has two crystals. Their retardance is controlled by an applied voltage (calibration is in fact setting these voltages). However their optical axes are fixed. One of them is at 45 degrees to the other. If the first one is parallel or perpendicular to the laser’s polarization then it is inert (or mostly so). By flipping it over, the first one went from being parallel to being at 45. Tremendously clear in hindsight!