ABSTRACT
While SMLM and STED discussed in the previous chapters bypass the diffraction limit by controlling the state of the fluorescent molecules (i.e., on/off), structured illumination microscopy enhances the resolution of optical microscopy purely in an optical way. This approach does not allow for the sub-100 nm resolutions of STED and SMLM but it nevertheless has several advantages. As SIM does not depend on the photocontrol of the fluorescent labels, it works “off the shelf” on a standard specimen without the need of special dyes, buffers, or cumbersome sample preparation. Historically the sample illumination is preferably homogenous, and much effort has been spent in the last 100 years to have a constant illumination over the largest field of view possible, from the Kohler condenser reported in 1893 [1] to the modern flat-field illumination used in SMLM [2]. SIM breaks this paradigm using a non-even illumination that generates a Moiré pattern between the structured excitation beam and the structured sample. After multiple acquisitions with different patterns, a mathematical reconstruction is applied and a super-resolution image obtained. In this chapter we will discuss the principle, the implementation, and the performances of SIM and present some representative applications in biology and material science.
