Practical Structured Illumination Microscopy

Abstract

Structured illumination microscopy (SIM) is a method that can double the spatial resolution of wide-field fluorescence microscopy in three dimensions by using spatially structured illumination light. In this chapter, we introduce the basic principles of SIM and describe in detail several different implementations based on either a diffraction grating or liquid crystal spatial light modulators. We also describe nonlinear SIM, a method that in theory can achieve unlimited resolution. In addition, we discuss a number of key points important for high-resolution imaging.

SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy

“…This is achieved by moving high-frequency information into the observable range of the microscope by frequency mixing with a fine-striped pattern of illumination. By imaging variations of the phase and angle of the illumination pattern this information can be computationally separated and recombined to reconstruct a super-resolution image with twice the frequency support in two or three dimensions 13 . Currently, the best practice in acquisition and processing of SIM data requires considerable expertise 14 15 which represents a significant barrier to exploiting the full potential of SIM imaging 16 .…”

Oxygen Tension and the VHL-Hif1α Pathway Determine Onset of Neuronal Polarization and Cerebellar Germinal Zone Exit

“…Images were acquired with the Slidebook software package, using a custom-designed 15 mm-square light-sheet pattern in the 5-phase structured illumination z-galvo and objective scan mode, with both colors being captured at each z position. The acquired images were background subtracted, then high-resolution SIM images were generated using an open-source, GPU-accelerated SIM reconstruction software with freshly acquired optical transfer functions (OTFs) generated on each day of imaging (Chen et al, 2014;Rego and Shao, 2015). Weka-mediated machine learning in Fiji was then used to analyze the numbers of LASP1-Emerald focal adhesions in maximum-intensity projections of LLS-SIM 3D volumes to determine the effects of Zeb1 or Hif1a overexpression on integrin-mediated adhesion.…”

Navigating challenges in the application of superresolution microscopy

“…SIM probably receives the most criticism for susceptibility to artifacts because of its strict dependence on computational reconstruction algorithms (Sahl et al, 2016). Indeed, great care must be taken during SIM system calibration and data acquisition to minimize reconstruction artifacts due to multiple possible sources: low SNR, background, mismatch between aberrations in raw images and the optical transfer function used for reconstruction, sample motion or photobleaching during acquisition, and illumination pattern inconsistencies across phases and rotations (Gustafsson et al, 2008;Rego and Shao, 2014;Demmerle et al, 2015). Low SNR or high background can manifest as spurious modulations of intensity (honeycomb) at spatial frequencies similar to the illumination pattern (Fig.…”