Super-resolution Optical Microscopy with Structured Illumination

Abstract

In widefield fluorescence microscopy, resolution improvement can be achieved by illuminating the fluorescent sample with a spatially modulated excitation source. The spatial modulation of light has been used in microscopy, in one form or another, for decades and has been exploited in both emissive and non-emissive modes. Techniques such as confocal microscopy and Hoffman contrast modulation microscopy can be considered as relying on the spatial modulation of light. Stimulated emission depletion microscopy also uses spatially modulated illumination to achieve super-resolution fluorescence imaging. Resolution improvement by structured illumination is based on Moire fringes. Unlike optical sectioning structured illumination microscopy (SIM) where the final image is calculated from three images, more images are required for isotropic resolution improvement with SIM. Successful reconstruction of a SIM image depends on the precise knowledge of the illumination structure and the imaging conditions. Photo-toxicity, particularly relating to biological samples, can impose severe limitations on the application of any optical microscopy technique.