Resolution limits imposed by classical light diffraction represent a major limitation in the ability to obtain meaningful insights into the fabric of living cells. Conventional optical microscopy tools have only revealed structures measuring approximately 200nm across. Unfortunately, most cellular organelles involved in physiological processes including cell-to-cell communication, cell growth and division are often below this limiting threshold. In fact, many cytoskeletal assemblies can be smaller than 50nm and our understanding of their function could be greatly improved if direct optical access to that nano realm was readily available.
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Light microscopy has always been suffering from the limit imposed by the Abbe’s law which manifested itself as presence of Airy disks. These structures defined the resolution of any optical systems regardless of imaging modality. For all users of microscopy systems it meant that image objects separated by distance roughly equal to half the wavelength of light used for imaging, coalesced into one diffuse spot. Until recently, the Abbe limit seemed to be the ultimate barrier in optical resolution therefore all images obtained from high performance light microscopes were irrevocably diffraction-limited. All finer structures in low sub-micrometre space were impossible to render without major distortions. To address this problem several novel imaging techniques have been developed to overcome the Abbe limit and to open up new avenues of research at nanometre scale.
Structured illumination microscopy has proved to be an excellent alternative to several existing microscopy techniques. Extending the capabilities of classical multi-colour fluorescence wide-field or confocal microscopic systems, it can provide sufficient resolution needed for analyses of fine intracellular structures like nuclear pore complexes or NPCs. These complex protein complexes are situated in the envelope surrounding cell nucleus and provide exchange pathway for number of molecules. NPCs mediate passage of RNA, ribosomes and signal molecules through the nucleus–cytoplasm boundary. This process assures continuous flow of information required for synthesis of proteins inside the living cell. Direct observation of NPC complexes with structured illumination microscopy allowed for the first time to perform series of analyses of subcellular structures beyond the diffraction limit and opened new routes to unravel new features of cell nucleus superstructure.
In the past few years we have witnessed an unprecedented evolution of imaging techniques, directed at helping researchers break through what was previously regarded as an immutable optical resolution limit. Several novel super-resolution methods have made it possible to look beyond ~200 nm into the realm of true nanoscale environments. These breakthroughs have been fuelled by the exponential growth of biophysical studies that often called for improved methods, required for precise localization and tracking of single labelled molecules of interest. As such, use of several cutting-edge single molecule fluorescent imaging techniques has made it possible to expand our insights into previously inaccessible nanoscale intra-cellular structures and interactions.
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