Today’s title isn’t as catchy as War and Peace despite the alliteration. Oh well.

Thanks to our finding a stable power meter, Dean and I were able to calibrate the liquid crystals on the multiphoton microscope. We made a little sculpture surrounding the detector and the rotatable polarizer (Fig. 1). The detector of the power-meter is a lightweight shaft with the sensor out at the end. This needed to be kept still. The polarizer has to perch on top, co-planar, and steadily enough that I can rotate it as needed from 0, to 45, 90, and 135 degrees. To fashion a platform for the polarizer, we stacked microscope slides on either side of the detector. The sculpture was stable. We calibrated, finding clear minima for all settings. 

When we started to image, the fun (and yes, I mean the “fun”) began. We were imaging celery xylem stained with Calcofluor. Because the xylem ribs are large, stain brightly, and are known to contain cellulose running parallel to the rib, they are a good standard sample. On the confocal previously, I imaged this material stained with fast scarlet or congo red. But now on the multiphoton, the screen was blank. The equipment guts are complex, many bits of hardware and software to set. Dean carefully went thru feature after feature; eventually, he discovered a wrong optical element inserted in the system somewhere. This explained why the total power during calibration had been so low; we were getting only a few percent of the expected power onto the sample. With the correct element in place, at last there was a signal. 

The fun got better. The signal, tho at least present, was not good. At first, I could not see any xylem cells. OK, time to get a little more specific in terminology. I have been using the term ‘xylem cells’ loosely to mean one specific kind of xylem cells: the water-transmitting cells (aka tracheary elements). Those are the cells whose walls are bulked up with cellulose-rich ribs, making them strong enough to withstand the tension exerted to pull water up to the leaves from the ground, and making them ideal test objects for my project. But xylem is a tissue; the tracheary elements are surrounded by strands of parenchyma, rank and file cells with standard-issue cell walls. When I dissect out the xylem from the leaf stem (aka petiole), the ensheathing parenchyma comes too. 

I could see plenty of brightly stained parenchyma. Only after some increasingly exasperated searching did I discern the tracheary elements: I had missed them because they were stained dimly. What the actual F? With fast scarlet or Congo red, the tracheary element ribs are blazing, and the parenchyma is evanescent in the background. Just the reverse with Calcofluor. I can hypothesize that the lignin found in the ribs repels the dye; but that it should repel Calcofluor specifically stretches my credulity. Regardless, I am in search of an alternate standard sample.

We might have tried a bunch of xylem-rib imaging anyway but the other problem we face is coordinating Micromanager and the multiphoton software. Micromanager controls the liquid crystals and the multiphoton controls everything else. To image a given field, Micromanager has to cycle the liquid crystals thru their settings in between the multiphoton acquiring and saving images. To our surprise, the multiphoton acquires and saves an image at the same time! Well, it starts saving well before it has finished acquiring. Dean and I wrestled with a workaround and Dean thinks he has one: But it requires writing a batch file to run some helper manipulations. 

We ended our session there. Next time, with the coding sorted, we will tune up the calibration at full power and image a different kind of standard sample and maybe try xylem ribs again. Step by step.