Conventional optical microscopes have a resolving power limited by the diffraction limit, first described by Ernst Abbe in the 19th century, which is approximately half the wavelength of the light used. Although this restriction makes it impossible to visualize objects less than about 200 nm, organelle membranes, cytoskeletal filaments, and protein complexes are all found below this size. It was only in the early 2000s that Gustafsson and others introduced Structured Illumination Microscopy (SIM), which overcomes this limitation by encoding high-fidelity spatial data into the image being studied by using patterned illumination.
Principle of Operation
The phenomenon behind the SIM working principle lies in the fact that the patterns of known illuminations interfere with the fine structural detail of the specimen and lead to the formation of Moiré fringes that hold the sub-diffraction spatial information. A sinusoidal grid pattern is normally projected on the sample due to varying angles and phases. SIM can be used to effectively increase both lateral and axial resolution by combining data from more than one orientation. The advantage of the technique is that it can utilise the response of linear optical sources, which enables it to be utilised with common dyes and fluorescent proteins, unlike nonlinear techniques, such as STED or PALM/STORM.
