SIM | SIM
Key Words: Super-resolution microscopy, STORM; Perfect Focus System (PFS), fluorescence
Definition:SIM is a super-resolution fluorescence optical microscope imaging technique that increases resolution by exploiting interference patterns (moiré patterns) created when two grids are overlaid at an angle
In SIM imaging resolution is increased by exploiting interference patterns (moiré patterns) when two grids are overlaid at an angle. Moiré patterns can be distinguished under the microscope, even when one or both of the original ‘grids’ is too small to resolve. In SIM, one of the grids takes the form of structured excitation light (essentially strips of light similar to a barcode), while the other is the specimen’s unknown distribution of fluorescent probes. As the pattern of light illumination is structured and, therefore, known, it is possible to obtain information about the pattern of fluorescence in the sample from the moiré pattern, which can be used to create both 2-D images and 3-D high-resolution sample reconstructions.
Comparison of conventional (left) and 3D (1 layer shown) N-SIM (right) imaging of YFP-labelled microtubules in B16 mouse melanoma cells.
SIM is able to resolve nanoscale cellular structures, for example, individual actin filaments and the inner membrane system of mitochondria. By accelerating the delivery of structured light patterns, SIM imaging can be applied successfully to living, moving specimens enabling the observation of, for example, tubulin and kinesin dynamics in living cells.
Nikon’s SIM imaging system (available through a license agreement with the University of California, San Francisco Office of Technology Management for Structured Illumination Microscopy) is based on the Eclipse Ti inverted research level microscope with Perfect Focus System (PFS) and CFI Apo TIRF 100x oil objective lens (N.A. 1.49). When used in TIRF-SIM mode, higher resolution TIRF observations are possible to give more detailed structural information near the cell membrane as well as providing the fastest imaging capability in the industry with a time resolution of 0.6 sec/frame enabling the capture of rapid cellular events. Suitable for both fixed and live cell imaging, SIM effectively doubles the resolution of traditional light microscopy and is of great value in structural, developmental, and intracellular communication studies. No special sample preparation is required enabling N-SIM imaging with normal single and multiprobe specimen preparation protocols.