I do a lot of work with electron microscopes. This is amazingly neat stuff, every bit as fun as I imagined it would be, and perhaps the closest thing that biologists have to being astronauts. I get to play with exotic, highly expensive equipment, and I get to see alien things that nobody has ever seen (or, in some cases, dreamed of) before. Twice in the last six months my lab has had a visiting scientist come to do work that is right up my alley, and since I am checked out on the microscope and she is not, I got to "drive" (the term that we seem to use for operating the 'scope) with her giving me instructions. It was very illuminating, seeing what is pertinent to our study and what can be skipped over, and at the same time there were several moments of both of us sitting and gaping and asking each other, "What on earth is that?!" It is fantastic stuff.
There are several kinds of electron microscope. The one that my lab uses most is the transmission electron microscope (or TEM), which operates in a very intuitive way: it works just like a regular light microscope, except that instead of shining a beam of light through the specimen, it shines a beam of electrons. The electrons are focussed using lenses, and (just as with the more expensive light microscopes) the images are captured by a camera. Of course, there are other significant differences as well: the lenses are in fact magnetic fields, and because electrons do not travel very far through air, the inside of the microscope is pumped down to a high vacuum.
Specimens for a light microscope are generally mounted on slides. This means that the specimen, usually aqueous in nature, is dropped onto a glass slide, over which is placed a coverslip. If the specimen is important enough, and stable enough not to degrade on its own, the slide may be sealed using Vaseline or fingernail polish, which can preserve the specimen indefinitely, but otherwise, that tends to be it. Of course, there are often also specific ways of preparing specimens, but I will not get into that right now.
Specimens for a TEM are analagous. The specimens are mounted not on slides but "grids", which are little (3 mm diameter) copper circles with a slot-shaped hole in the middle. (Yes, they are not actually gridlike: this is a historical term, used because other grids actually do have mesh gridwork in the holes. Many labs use those kinds as well, but my lab has so far used only the other kind, called slot grids.) The specimen is actually mounted on a very thin plastic film, on the order of 40-100 nm thick, which is suspended in the middle of the grid. Like a well-preserved slide, the specimen will last indefinitely.
There are of course other differences, many of which are obvious upon contemplation, and some of which require further explanation (which I may get to in other posts). However, one difference that is significant here is that electron microscopes (all of them, but particularly TEMs) are not just sending a beam of light through the specimen: they are shooting charged particles, which can and often do interact with the specimens. Electrons can be really harsh! In the case of my recent work, for which I was using very thin films (40 nm or so), the electrons can rip right through the specimen. This means that -- often right after discovering the perfect example of a cell amongst dozens of unusable contenders -- one can watch one's precious work tear apart, wrinkle, and wither away before one's eyes. No amount of preparation can prevent this: it just happens. Thicker films are less prone to this sort of damage, but they also impede the electron beam more, and so lose resolution. Resolution is the reason why we use electron microscopes in the first place, so we tend to use the thinnest films that we can. Obviously, I need to experiment more with this, to find a better film thickness that will not break apart in the electron beam, without losing too much resolution.
There are numerous other ways in which TEM specimens can be irreparably lost. One of the most frustrating is tweezering -- putting a hole in the film with clumsily handled forceps while moving the grid around. I have had much hapless experience with that. Another is that some of the stages (the devices that hold the specimens inside the microscope) hold the specimens in place with spring clips, which can and often do tear through the films when removed (or, on occasion, when put in). There are ways of minimising this, which I have only recently become competent at.
Unfortunately, "recently" means that I had lost several specimens while learning how to handle, mount, and dismount them. Up until last Friday, I had only one pristine specimen left, undamaged by my apprentice clumsiness. That specimen, last Friday, self-destructed in the 'scope, as the film gave way spontaneously. This is a setback, of course, but I can always make more specimens. This is of course how science works. Only now, I will try thicker films!
Monday, 25 February 2008
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