Sunday Jan 11th Back to the lab

In which is recounted the bright side of working in the dark

No, I cannot claim to have spent all week astride my bench, nor even most of it. I had a couple of annual reports due for grants, another one for UMass administration (yes they keep a beady eye on their profs), a review for Science, and a 600 word manuscript summarizing my issues with that paper on auxin binding protein 1 (see earlier posts, Dec 14th and 21st). But all the same a little lab progress was made. Enough so that I can leave aside polemics, philosophical and scholarly, and focus on the experiments.

My attention turns to the segment growth system. I have described this project in earlier posts (for example, 31/8 meniscus; 14/9 diversity; 26/10 futzing-second half), but even so I will probably repeat things a bit here to save you (and myself!) from fossicking around through LabFab back issues. Setting up this system has required glorious tinkering because I insist on working in total darkness. Not because I think that is the ideal metaphor for the human condition (cynics!) but for reproducibility. The project is all about growing stem segments, objects that grow sluggishly in the light but spritely in darkness. Fast growth means better experiments (easier to measure the growth and a larger dynamic range–considering that most treatments inhibit growth, the absolute amount of expansion sets the scale for the largest measurement).

Now, I would have already gotten plenty of data were I willing to work in dimness. But the problem is that these stems are ridiculously sensitive to light. This means that even the various LEDs twinkling on equipment, the light coming under the door, and certainly the light from the computer monitor will affect the plant’s response. Moreover, dimness is difficult to measure, typical light meters don’t go down that far. How do I record the total load of dim light sources? Not possible. But darkness is reproducible. Well, of course, like absolute zero, darkness is an impossible ideal–there will always be a few photons in any black box. My criterion is no photons that my eye can detect after dark-adapting in the black box for half and hour.

It is the requirement to do it in the dark that gives rise to all the futzing. I will measure growth by taking images of the segments, at defined times as they grow, with a video camera interfaced to a computer and imaging in infrared. As an aside, imaging is the key innovation that I hope will allow me to do better experiments than those previously because most of the earlier work relied on position-transducers, electrical gadgetry that had to be glued onto the segments. Imaging has the twin advantages of avoiding mechanical disruption and allowing growth in both length and width to be measured.

Anyway, this week, I ran wires through wall so that the computer can be outside the experimental chamber. To get to the computer to capture images, I will have to open and close the chamber door (which fortunately seems totally light-tight). Therefore, the computer has to emit no light at all while the door is open. I have taped up all of its LEDs and installed a cute tool that that toggles the monitor on and off with a key stroke. I also found a swag of black cloth to put at the base of the door to the corridor to block off the light leaks there. Just above the handle on the front side of the door to the corridor, I put a hook and made a sign that says “Caution—plants growing. Please minimize corridor light” on one side, and “STOP! Experiment in progress. Do not open the door.” on the other. The “stop” is in a red octagon and the “caution” is in a yellow triangle – the sign was fun to make. This has to be done because my experimental chamber is one of three rooms serviced by a small corridor. The other rooms are accessed occasionally.

This week, I also finished assembling a set of six segment holders. Each one has a Teflon washer glued onto a 4 inch by 3 inch (roughly) glass plate. The inside diameter of the washer is about 20 mm and it is perhaps 2 mm deep. One mL of solution fits nicely and is perfect for floating segments, with little trouble from the meniscus (thanks Teflon!). The plates are recycled from a no-longer used apparatus for mini-gels. Darren built a tower out of Plexiglas, that straddles the video camera and has a shelf on which the glass plate rests. I placed heavy backstops on the sides and back of the tower, so I can put the segment holder plate in and out and hit the same spot every time. The camera is focused so that the inside of the washer fills the field, showing just a whisper of the washer at the edges of the field. This ensures as many pixels as possible per segment, with nothing getting lost outside the field. I had to glue the washer carefully so it would occupy exact same spot. One of them missed but by an amount small enough to accept as a cost of doing business. Now, I can have up to six treatments per experiment. If I need more, I’ll have to scrounge more glass and get the glue out again.

The top of the tower has a few sheets of thick tracking paper that serves to diffuse the light from the LED light source that is positioned 15 cm or so directly above. This lights the field of view evenly. I had been testing this set up for some time and feel that it is all it can be, at least for now. Sorry I haven’t thought to snap a picture. Next time.

The remaining tasks deal with my cheating eyes. Oh, I guess it is my heart that is supposed to be cheating and my eyes that are lying. Hmmm —maybe time for a new song? What I ought to do, and what my pals Jim Shinkle and Dina Mandoli among others did in Winslow Briggs’ lab in the glory days of phytochrome research was to suck it up and work in total darkness. But I am just too old. As I think I have mentioned before, I am going to use an device that converts infrared to visible so that I can see something. Yes, that means my segments will see infrared light. The good news is that seedlings are blind to this kind of light, at least at doses characteristic of illumination (not heat). The device is sold by an military store (hence ‘device’ is an apt word) and is a monocular, about the size of half a pair of decent binoculars. I also bought a harness that holds the device on my head. I could call the harness a ‘helmet’ except it is not so helmetish, having instead just a few bands and straps that cradle the head.

This week, I practiced using the device. I will admit to being scared. The only time I had actually used one of these things was in 1985 when I was working at Okazaki, Japan, as part of my Ph. D. The lab there had one in the photographic darkroom, for loading film onto racks for development. I had gotten really good ! at doing this by feel in the dark but they had a ‘sniper scope’ to let you see your fingers and the film. I tried it just once – it made a high pitched whine that hurt my head and it was so disturbing seeing the film that I preferred the darkness. Fortunately the device I ordered here emits no sound (or I have lost the relevant part of my hearing). But how tricky will it be in operation? A blurry awkward mess?

Well, I put the thing on my head, turned off all the lights, and did a kind of dry run, including cutting some sections. While I would not say it was just like working in the light, it was not so bad. I get just one eyeful, and the focus is a bit farther from my head that I might wish. Still in all, doable. This came as big relief.

There is a problem though –visible light comes out of the eyepiece. Some of it surely will bounce off my face and onto the plants. To minimize this, I cut a piece of black cloth to serve as a mask. Then, I removed the device and brought harness and cloth home to Laura, the seamstress I know and love. She sowed the cloth onto the harness in such a way that it drapes over my face, and cut a slot so I can reattach the device. I’ll need also to cut an eye hole. And while this might allow a green photon or two out, I think not so many. I can check with my other eye!

Actually, one more problem. The maze seed that I set up one Monday so I could do this device practice thing had little more than germinated. In earlier months, that was enough time. There is no temperature control in the room but I do have a temperature data logger. I need to get the readout and see if I can account for the delay based on cooling of the room. Winter? I also have to find out if storing maize seed at room temperature impairs vigor? I think it might. Still I can always allow more time or if the temperature in the room turns out to vary like a drunk walking home, I can grow the plants elsewhere as long can they can be trebly swaddled to keep them dark. We’ll see.

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