Chapter 17 - Structured Illumination Imaging

Abstract

A structured illumination microscopy (SIM) setup is the one in which the specimen is illuminated with a specific spatial pattern rather than with uniform illumination. The most common use of structured illumination is in optical sectioning microscopy. Linearly sinusoidal SIM is a fast optical sectioning tool that requires only simple modification to the conventional light microscope followed by a bit of image processing. A mechanical actuator attached to a linear sinusoidal grating is used in place of the grid with a coherent light source. The sectioned image should be computed with high numerical precision (e.g., double-precision floating point). Then one can apply further image processing if necessary. If so, high precision should be used there as well. The values resulting from the floating-point calculations must be scaled to the integer range of the final output file. For example, for 8-bit precision, the overall range would be scaled from 0 to 255. But this is not completely straightforward. Usually a series of optical sections is gathered at regular intervals along the z-axis to cover a three-dimensional specimen fully. The entire set of images must be considered when scaling gray-level values, since all sections should be scaled consistently.