Superresolution Multidimensional Imaging with Structured Illumination Microscopy

Abstract

The resolution of an optical microscope is fundamentally limited by diffraction. In a conventional wide-field fluorescence microscope, the resolution limit is at best 200 nm. However, modern superresolution methods can bypass this limit. Pointillistic imaging techniques like PALM (photoactivated localization microscopy) and STORM (stochastic optical reconstruction microscopy) do so by precisely localizing each individual molecule in a sample. In contrast, STED uses the stimulated emission process driven to saturation to dramatically reduce the size of the region in the sample that is capable of spontaneously emitting fluorescence. Structured illumination microscopy (SIM) illuminates the sample with a pattern, typically the image of a grating. This computationally removes the out-of-focus blur, a method known as optical sectioning SIM. Furthermore, frequency mixing of the illumination pattern with the sample caused by the moiré effect results in a downmodulation of fine sample detail into the frequency-support region of the detection optical transfer function. High-resolution SIM achieves typically a twofold lateral resolution enhancement. This is further improved by exploiting a nonlinear sample response to the illumination light in SIM. Recent developments of the method allow fast, multicolor, and three-dimensional high-resolution live-cell imaging.