Nanoscopy

The Nobel Prize in Chemistry for 2014 has been awarded to Eric Betzig, Stefan W. Hell and William E. Moerner for the development of super-resolved fluorescence microscopy. This technique allows scientists to view objects at the nanometre scale and is therefore referred to as nanoscopy.

Since the 17^th century, we have been able to peer into the world of very small things using optical microscopes. In 1873, microscopist Ernst Abbe published an equation to show that optical microscopes could not be used to investigate things that were less than half the wavelength of light, that is, to be seen in an optical microscope the object must be greater than 0.2µm. An optical microscope can therefore be used to see some surface structure of a human hair, but you couldn't use it to see the actual protein building blocks making up the hair.

Stefan Hell was working on fluorescence microscopy, using fluorescent molecules to image parts of a cell. A brief pulse of light makes the fluorescent molecules glow temporarily, following the glow allows scientists to map where the molecules are in the cell. The technique can be used to tell where DNA is located for instance, but it could not be used to determined its structure. Stefan Hall proposed a new method, Stimulated Emission Depletion (STED) in which one pulse of light excites all the fluorescent molecules while another pulse quenches the fluorescence from all the molecules except those in a nanometre-sized volume in the middle. Only this volume is registered. An image is built up be sweeping along the sample and continually measuring light levels. In 2000 Stefan Hall was able to demonstrate the effectiveness of the STED microscope by imaging an E.coli bacterium at a resolution that could never be achieved using an optical microscope.

The nanoscopy method proposed independently by Eric Betzig and W E. Moerner, Single-Molecule Microscopy differs in that it relies on the the superposition of several images.

In 1989, W E. Moerner measured the light absorption of a single molecule for the first time.
W E. Moerner had found that one variant of green fluorescent protein (extracted from fluorescent jellyfish) could be made to fluoresce with light of 488nm wavelength, but that after awhile, the fluorescence faded and would not fluoresce again using 488nm light. The same protein, when hit by light of wavelength 405nm could be brought back to life, and then would fluoresce again when hit with light of 488nm.

In 2006 Eric Betzig demonstrated the usefulness of Single-Molecule Microscopy using a glowing protein coupled to a cell's lysosome. Using a weak light pulse, only some of the molecules were caused to fluoresce, and these were at distances greater than 0.2µm. This image was registered. When the fluorescence of these molecules died out, a new weak light pulse was used to initiate the fluorescence of a few more molecules.This new image was registered. This process was continued many times. When Betzig superimposed all the images, a super-resolution image of the cell's lysosome membrane was the result.