MBL reprise

Tobias redivivus. Besides sprightliness, I also feel chilly, sitting at picnic table waiting for the laundry machines to finish. One of the pleasures of being here is indeed the fresh ocean breezes, moderating the temperatures. Even better, the ocean in question is 50 yards from my office window and delights the eye even when the rains fall.

I am here because, happily, I secured a Whitman Center Fellowship, allowing me to spend the summer working with MBL scientist, Rudolf Oldenbourg. If you are regular reader of LabFab, this will sound familiar: I was here last summer, working with Rudolf, and I posted eight times, starting here.

I have spent the last two weeks setting up stuff, mostly. I need to grow BY-2 cells, the objects of my summer study. These cells grow as single cell filaments in liquid culture. In that, they resemble algae, but in fact they originate from a tobacco plant and have been growing in liquid culture since the 1960’s. Growing them requires a shaker that can hold a constant temperature. Happily MBL has such a unit available to loan, indeed I borrowed it last summer. The shaker was delivered and the platform even retained the hardware I hunted up last year to secure flaks. I covered the lid of the shaker with aluminum foil to keep the cells dark (Fig. 1). They are plant cells, which are supposed to like, even need, light. But not BY2 cells. They have lost the recipe for photosynthesis, even tho they retain plastids, the cellular organelle where photosynthesis would happen. When the cells are grown in the light, the plastids get … confused, and make overtures toward photosynthesis. Best to avoid inducing such metabolic mayhem. The shaker ran and appears to hold temperature just fine.

Figure 1. Shaker used to culture BY2 cells. The fresh coat of aluminum foil helps keep the light out.

I repaired some lab infrastructure. Rudolf has two rooms, one with his microscopes and another small room for bench work. This room has long been used to grow marine organisms, sea urchins or tunicates, in a large set of tanks that take up half a wall (Fig. 2). At the moment the tanks are empty and no one is likely to use them any time soon. The tank is next to small bench and a big lab sink. The bench was laden with salty apparatus, accumulated over the years of caring for and maybe harvesting the sea creatures. The sink was grimy and sandy and rusty. There was no distilled water for rinsing glassware. The cabinets and drawers hadn’t been organized in a decade. Last summer, I avoided this mess by using a neighboring lab to prep my glassware. But this summer the neighboring lab is going to be occupied and even tho Rudolf oversees the space (my lab key opens the door) and I would probably be “allowed” to use the room, I wanted to stay out of the new occupants’ hairs. I rolled up my sleeves and cleaned up the bench and the sink, organized the cabinets and drawers, and got an enormous carboy of distilled water aloft (thankfully the shelves above the sink are solid) rigged up to dispense clean water for rinsing.

Figure 2. Tunicates growing in the salt water tanks in the Oldenbourg lab, last summer. This summer, the tanks are empty.

I made growth medium, in an episode that was strangely reminiscent of Laura’s bad dreams, where one thing after another after another goes wrong. But in the end, the stuff was ready. Last weekend, I drove to Amherst, helped Laura pack, and on Monday morning,  got a confluent culture of BY2 cells from Eri, and returned to MBL.

During the week, I did a simple experiment, designed to provide ‘baseline’ information. A few hours after depositing the cells on the shaker, I sub-cultured them and set up a replicate flask to sample. This was supposed to take ten minutes but took the better part of an hour because the trusty ethanol burner refused to light. I ran thru two matchbooks and much cursing before finding a different burner. I sampled the culture on the following four days, each day imaging cells with transmitted polarized light microscopy. In other words, there was no experimental treatment, just look at the cells at each day of their culture period. I imaged about 50 cells per day, and will measure the birefringent retardance of their cell wall to see whether this parameter varies during culture. I picked up a hint of variation last summer in the controls for one experiment. Also, this simple experiment was a good way to remind myself how to use Rudolf’s set up. I did need a few trials to get in the groove as it were, there being many things to get right. I won’t know how this turns out until I do the measuring, sometime next week. But at least the cells appear to be growing well (Fig. 3).

Figure 3. Shaker with the lid open and the platform motionless. The two flasks have nearly confluent cultures of BY2 cells.

Inter alia, I had an amazing demonstration of the power of ‘structured illumination’. On the second day of imaging, the high powered arc lamp was flickering. While this lamp is essential for fluorescence, it is not needed for retardance measurements with transmitted light. I could change over to the ordinary tungsten halogen lamp. But this kind of lamp requires a diffusing screen otherwise the lamp filament itself causes the light background to be hugely uneven. I popped the diffusing screen in and started to image. The results were horrible, as if my beautiful cells had been suddenly infected by a fungus and turned to mush. The revolting images turned to be explained by the diffusing screen having been burned. Rudolf showed me by focusing on it and it was a field of black spots, like a really bad sunspot year. In the ordinary focus position (looking at the rear focal plane with a Bertrand lens), the spots blurred out to a faint and more or less uniform loss of intensity. Happily, I could move the screen a tad, placing a non-burned position in the beam, and Hey Presto  images looked great again. The point is that by blacking out some regions of the incident light, the consequences for image formation are massive. There is a kind of microscopy that leverages this effect for good and now I can appreciate the power of the principle at play.

I also got trained on the confocal fluorescence microscope, upstairs in the core. I am hopeful that this will be a better instrument for my project than the widefield (i.e., ‘conventional’) fluorescence microscope in Rudolf’s lab (and which I used last summer). The confocal is a complex thing and the software to run it has more bells than a Pachinko parlor. Adding to the fun, the ability of the instrument to do polarized fluorescence is run by a homemade macro, the interface for which is a little bit … non-intuitive. But I got started. This week, I’ll image BY2 cells with it and see how well the instrument performs. In theory it should be wonderful, but in practice…