Through the looking glass - the adventures of seeing beyond the diffraction limit

Continuous developments have been made in the field of looking glass since Antoni van Leuwenhoek first invented microscope. Driven by the founder of cell biology, Matthias Schleiden, a Jena-based entrepreneur from Germany, Carl Zeiss made an effort to improve and systematically craft reproducible high-resolution lenses. For this purpose he hired in 1866 young physicist Ernst Abbe as a freelancer and asked him to systematically improve lenses by introducing a mathematical basis. He developed a theory of image formation in a microscope that led to the discovery of the resolution limit due to the wave nature of light, namely diffraction. It was, however, only in 1911-1913 that the well-known players in microscopy, Carl Zeiss in Jena and Carl Reichert in Vienna developed a fluorescence microscope that was commercially successful. In the late 1970s, Christoph and Thomas Cremer first came up with a path-breaking idea of using two oppositely placed objectives in a microscope to obtain interference in the focal plane, a so-called 4π technique in the confocal fluorescence microscopy that improved resolution as well as inspection of the depth of field. The 1990s also witnessed another approach to superresolution microscopy based on wide-field microscopy to image cellular nanostructures stained with fluorescent markers. It is based on the statistical fluorescence behavior of single fluorescence emitters known as 'blinking' that can be exploited for single-molecule detection.