Super-Resolution: A History of the Beginning, by Marc Toso

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No one doubts that light microscopy has come a long way since the 16th century. The first known compound microscopes were built by Zacharias and Hans Janssen. None of these microscopes survived, however they are believed to have boasted a magnification of 3X to 9X. It was Leeuwenhoek in the late 1600s that first pushed magnification and resolution to the cellular level. His surviving microscopes have a magnification of 275X and an impressive resolution of 1 micrometer. It was later in the 1800s that Ernst Abbe discovered that the resolution of an optical microscope is determined by the function of the wavelength of the incident light and the microscope's numerical aperture. This pushed the resolution limit to around 220 nm. It was at this limit where we microscopists remained for the next hundred or so years. The restrictions of this spatial resolution eliminated the possibility of resolving subcellular structures such as ribosomes, vesicles and other molecular interactions, which are all beneath the resolving power of light microscopy.

Electron microscopes came along not too much later in the 1930s from German scientists Ernst Ruska, and Max Knoll which pushed the resolution limit down to around 10 angstroms. This was an unbelievable feat.  It was not too many years later that biologists began taking advantage of this novel tool. In March 1945 in the Journal of Experimental Medicine Keith Porter, Albert Claude, and Ernest Fullam published the very first electron micrograph of a cell in their paper ‘A Study of Tissue Culture Cells by Electron Microscopy’. The following decades resulted in extensive experimental work in which the TEM revealed detailed and intricate ultrastructure of biologist.

However, the purpose of biological microscopy is to understand how cells function while alive. This "life" aspect was the price scientists needed to pay for the TEM's amazing resolution. Cell samples were required to be fixed, dehydrated, embedded in plastic and sliced into ultrathin sections. This resulted in a 2-dimensional image of a highly processed and ultimately extensively manipulated sample. We have come a long way finding suitable fixatives and buffers that minimize artifacts. Likewise, with ultra-thin serial sectioning three-dimensional reconstruction of cells is now a possibility, but the data still represent a snap-shot in time. Life is more than a snap-shot, life is dynamic.

Here is where super-resolution microscopy enters the field. To fully understand   dynamic life processes we needed to push through the resolution limit of light and apply modern techniques of fluorescence and confocal imaging to the sub-cellular level. We needed superior resolution that could be applied to living cells. In 1978 the two brothers Thomas and Christopher Cremer published 'Considerations on a laser-scanning-microscope with high resolution and depth of field' in Microscopia Acta. These observations opened the door to observations beneath the diffraction limit of light.

All of the major microscopy vendors, such as Leica, Zeiss and Nikon offer commercial super-resolution units where resolution down to 20 nm is not an uncommon claim. Even smaller start-up companies such as Vutara in Utah, which claims to have the first 3D super-resolution microscope, are emerging into the field. Live cell imaging at this resolution is now becoming commonplace and is found extensively in the literature.  

This 3D structured illumination microscopy (3D-SIM)-3 prophase image was taken by Lothar Schermelleh, from the paper "Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy" 

This super-resolution GFP image is by Christoph Cremer "Dual color localization microscopy of cellular nanostructures."Biotechnology Journal, 2009, 4, 927-938
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