Super-Resolved Fluorescence Microscopy: Surpassing the limitations of the light microscope

The Fluorescence Microscopy was currently in news as the 2014 Nobel Prize for Chemistry acknowledged the development of super-resolved fluorescence microscopy. Eric Betzig, Stefan Hell And William E. Moerner are the names behind the development.  

Eric Betzig of Janelia Farm Research Campus, Stefan W. Hell of Max Planck Institute for Biophysical Chemistry and William E. Moerner of Stanford University bypassed a presumed scientific limitation and proved that an optical microscope can yield a resolution better than 0.2 micrometres.

Development of Super-Resolved Fluorescence Microscopy:

•    Stimulated Emission Depletion (STED) method- Stefan Hell
Since 1990, Stefan Hell was looking for a way to bypass the limitation that Ernst Abbe had defined more than a century earlier. The thought of challenging such an established principle was tantalizing, but senior scientists in Germany had met his enthusiasm with scepticism. Hell was convinced that there had to be a way of circumventing Abbe’s diffraction limit.

Later, Hell worked on Fluorescence Microscopy, a technique where scientists use fluorescent molecules to image parts of the cell. He read about stimulated emission and realized that it should be possible to devise a kind of nano-flashlight that could sweep along a nanometre at a time.

In 1994, Stefan Hell published an article on Stimulated Emission Depletion (STED) method in which a light pulse excites all the fluorescent molecules while another light pulse quenches fluorescence from all molecules.

After studying the STED method, he developed a STED microscope. In 2000 he was able to image an E. coli bacterium at a resolution never before achieved in an optical microscope. The STED microscope collects light from a multitude of small volumes to create a large whole.

•    Single-Molecule Microscopy Method- Eric Betzig and W. E. Moerner
In 1989, W. E. Moerner measured the light absorption of a single molecule. At the time he was working at the IBM research centre in San Jose, California. The experiment opened the door to a new future and inspired many chemists to turn their attention to single molecules, one of them was Eric Betzig.

In 1997 W. E. Moerner discovered that the fluorescence of one variant of green fluorescent protein (GFP) could be turned on and off at will.

Moerner dispersed these excitable proteins in a gel, so that the distance between each individual protein was greater than Abbe’s diffraction limit of 0.2 micrometres. Since they were sparsely scattered, a regular optical microscope could discern the glow from individual molecules as they were like tiny lamps with switches. By this discovery Moerner demonstrated that it is possible to optically control fluorescence of single molecules.

Just like Stefan Hell, Eric Betzig was obsessed by the idea of bypassing Abbe’s diffraction limit. In the beginning of the 1990s he was working on a new kind of optical microscopy called near-field microscopy at the Bell Laboratories in New Jersey. In 1995 Eric Betzig concluded that near-field microscopy could not be improved much further. But Abbe’s diffraction limit remained in his mind.

Inspired by W. E. Moerner, Eric Betzig had already detected fluorescence in single molecules using near-field microscopy. He began to ponder whether a regular microscope could yield the same high resolution if different molecules glowed with different colours such as red, yellow and green.

His idea was to have the microscope register one image per colour. He demonstrated that if all molecules of one colour were dispersed and are not closer to each other than the 0.2 micrometres stipulated by Abbe’s diffraction limit, their position could be determined very precisely.

Later, when these images were superimposed, the complete image would get a resolution far better than Abbe’s diffraction limit and red, yellow and green molecules would be distinguishable even if their distance was just a few nanometres. In this manner Abbe’s diffraction limit could be circumvented.

Ernst Abbe’s diffraction limit
For a long time, optical microscopy was held back by a physical restriction as to what size of structures are possible to resolve. In 1873, the microscopist Ernst Abbe published an equation demonstrating how microscope resolution is limited by the wavelength of the light.

Resultantly, for the greater part of the 20th century the scientists believed that they would never be able to observe things smaller than roughly half the wavelength of light 0.2 micrometres in optical microscopes.

The contours of some of the cells organelles were visible such as the powerhouse mitochondria, but it was impossible to discern smaller objects like interaction between individual protein molecules in the cell.