Since my knowledge and understanding of neurobiology are rudimentary at best, I thought I would write a brief entry on the Nobel Prize in Chemistry instead. This year’s prize was jointly awarded to Eric Betzig, Stefan W. Hell and William E. Moerner “for the development of super-resolved fluorescence microscopy”. [A lot of the information in this entry came from here.]
Principally, one can distinguish between two types of super-resolved fluorescence microscopy: either when an ensemble of fluorophores (those chemical entities emitting light upon appropriate irradiation) is involved or when single fluorophores are being imaged. The former technique came into use in around 2000, while the latter has only been available since 2006; there seems to be a general trend towards shorter timespans between when the scientific discoveries/developments of a technique are made and when the prizes are awarded.
Why bother with super-resolved fluorescence microscopy in the first place? It’s such a mouthful that it might not be worth the effort to develop. However, if you have a look at the following image, entitled “Untangling Neurofilaments” and submitted to the Cell Picture Show by none other than Stefan Hell, then you might realise that it is a really useful technique [image copied from the Cell Picture Show website].
The image on the left shows neuronal filaments and was acquired using confocal fluorescence microscopy, a technique that is already more advanced than standard or epifluorescence microscopy because it only allows visualisation from a single focal plane, thus increasing contrast and resolution. On the right, however, the same filaments are imaged using a super-resolved technique called stimulated emission depletion (STED). Clearly, this second micrograph shows a lot more detail and one can distinguish different filaments from one another. This can be important when trying to distinguish pre- from post-synaptic neurons, for example.
The main problem in optical microscopy, as alluded to above, is that of the diffraction limit: two objects that are closer to one another than approximately half the wavelength with which they are being visualised cannot be distinguished. For example, when imaging with blue light (the light with the shortest wavelength before it becomes UV light) two objects closer than 200 nm (400 nm divided by 2) will appear to be a single object. And this is the theoretical limit; in practice the resolution is worse. [For a more rigorous definition of the diffraction limit and a more physics-based/mathematical discussion I would recommend the “Scientific Background” provided by the Royal Swedish Academy of Sciences.]
A typical bacterial cell is about 2000 nm by 500 nm and so by conventional light/fluorescence microscopy these cells can be visualised, but their internal structure cannot be resolved. Super-resolved fluorescence microscopy relies on visualising only a subset of all fluorophores in a given sample and being able to pinpoint more precisely from where the photons are being emitted (the physical explanation of this, I’m afraid to say, is beyond my capabilities; best to check here and in the references there, if you are interested).
However, something I can explain links perfectly back to one of my favourite classes of proteins, the fluorescent proteins (FPs). When William Moerner was studying green fluorescent protein (GFP) mutants from Roger Tsien’s lab (Nobel Prize in Chemistry 2008), he noticed that some of them had to be activated before they would fluoresce at all and could be irreversibly turned off as well. This allowed the switching on of only a subset of FPs and the detection of these at super-resolution. Subsequently, this first subset is switched off and the next subset turned on, so that sequentially all FPs can be imaged and the whole picture put together. The method is called PALM (Photoactivated Localisation Microscopy).
Just like with the CRISPR Craze, I first heard about these microscopy techniques last year in a supervision: one of the M.Sci. students was presenting her project which involved PALM imaging of an enzyme as it moved along DNA; to control for the fact that the DNA itself might be moving she tracked a histone variant (histones are proteins that associate with DNA to facilitate its tight packing) and used it as a proxy for DNA movements.
Lastly, although this intellectual and technological achievement certainly deserved to be recognised by a Nobel prize (but why the Chemistry prize I’m not quite sure – Medicine/Physiology or Physics actually both seem appropriate too), I wonder how “right” or “fair” it is to award these prizes to individuals. On the one hand, yes, these three men all had great ideas about how to improve light microscopy, but on the other hand, they presumably had a tremendous amount of help from their lab members. Furthermore, science today is far more collaborative than it was when Alfred Nobel lived. I wonder whether there is a way to make sure more people are acknowledged for their hard work…
Oh, and have I mentioned that I love cells? If you do too, then maybe one of the activities at Biology Week will tickle your fancy.