I remember one of the best presents I ever got as a kid was a microscope. I have no recollection whatever of who it was that got it for me but I remain grateful for it to this day. Unfortunately my successes at imaging the smallest structures in biology were few, in fact they were non-existent. I don't remember managing to get a single sample to work. I didn't know that just looking at
pond water or peeling
an onion could yield such impressive results so easily. Mainly, then, my microscope remained in its box looking at nothing more interesting than the back of the instruction manual; but it wasn't as fruitless a situation as it sounds. Even though I couldn't use it properly I loved
having it. It inspired me. I liked that someone would entrust me with this highly sophisticated piece of scientific machinery, which is what I assumed it was. The fact is, though, that microscopes aren't especially sophisticated, at least not your standard compound microscope that most of us would be familiar with from high school biology classes. It isn't entirely clear who invented it, or when, but if we said 400 years ago we wouldn't be far off the mark, though it wasn't until the 1670s that microscopy became recognised as a way of seriously studying nature as opposed to merely a gentleman's hobby.
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| "...green Weeds growing in Water and some Animalcula found about them" by Antonie van Leeuwenhoek. |
Native of Delft
Antonie van Leeuwenhoek became the stand out practitioner of the field. He was a slightly odd man who never actually published scientifically or shared the details of how he achieved his progress; he nevertheless came to dominate the field to such an extent that Robert Hooke commented he had a virtual monopoly on microscopic study and discovery. Leeuwenhoek was the very first person to see images that we now commonly take for granted. Bacteria, the cell vacuole and, ahem, sperm were all amongst his discoveries. He also coined one of my all time favourite words: animalcule. He used this to refer to the bacteria and other microorganisms he was discovering. It is only through his correspondence with the Royal Society in London, who subsequently published both his letters and accompanying diagrams, that we can see the incredibly detailed work he was able to carry out.
As we learned more about the nature of light itself it became clear that we would never be able to see anything smaller than about 200-250nm; it's just not possible. There is an equation that Ernst Abbe (a contemporary of Carl Zeiss, camera fans!) came up with in 1873 which I don't fully understand but the result happens to be approximately half the distance of the wavelength of light. For a century this remained unchanged and unchallenged but then some clever people decided to take a different tack which would improve resolution ten fold. And make some pretty pictures too.
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| Super high resolution image of an E. coli cell by Eric Betzig |
Two methods were independently developed both of which have pretty cool acronyms (actual acronyms, not just initialisms): photo activated localisation microscopy (PALM); and the closely related stochastic optical reconstruction microscopy (STORM). It was Eric Betzig, of the Howard Hughes Medical Institute, that developed the PALM technique, so to speak. I don't fully understand how the technique works but it starts out similarly to standard fluorescent microscopy. A fluorescent molecule is tagged to the molecule of interest either using an immunohistochemical approach (antibodies) or genetically. Then the trick is to repeatedly switch the fluorescence on and off such that you obtain a high contrast between your illuminated molecule of choice and the background; subsequently you need to apply some cunning Gaussian functions to help localise the actual centre of the diffraction blur. This is made much easier if you have a small number of areas being activated at any one time and if these points are giving off a large number of photons.
Above you can see an image of a cell from a fruit fly,
Drosophila melanogaster. On the right is the traditional image you could expect to get using normal microscopic techniques. On the left is the same cell using the PALM technique. You don't need to be a cellular biologist to see that there is a lot more detail to be seen using the new technique. Indeed, the image is actually 3d as the different colours represent different depths within the cell. The spaghetti like structures are the microtubules of the cell which are a sort of scaffolding that holds the whole thing together and is also very important during cell division.
In this remarkable image the same cell has been represented 3 times. In it, the mitochondria, the energy producing organelles, have been made to glow. In the left panel is the image you would expect to see using standard techniques. The right hand panel is a single cross section through the cell using the STORM technique and in the central panel is a super resolution 3d image using STORM where colour once again represents depth. One of the differences between PALM and STORM is how they label the molecules of interest. PALM uses the genetic method and STORM uses the immunohistochemical approach. There are a couple of differences to do with the actual imaging but frankly I don't understand them well enough to be able to explain them.
So asides from creating beautiful images and winning Nobel prizes, what are these new methods actually good for? Well, one exciting example was announced on the same day as the Nobels. Researchers were able to show how HIV changes its shape as it invades a T-cell thereby opening up another new avenue to explore for possible treatments or vaccine developments. This is very important work and shows science at its most creative and beautiful.
