Abstract
Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.
Highlights
Over the last half a decade, super-resolution microscopy has matured and moved from specialist optics labs into the hands of the biologist
The point spread function (PSF) of a microscope is well approximated by the image of a single sub-diffraction sized fluorescent bead, the PSF and the resolution can be quantified by fitting 2D Gaussian functions to individual beads for each wavelength
Custom-built total internal reflection fluorescence (TIRF)-structured illumination microscopy (SIM) systems such as the setup detailed in this protocol are capable of multicolor super-resolution imaging at high speed compared to commercially available microscopes
Summary
Over the last half a decade, super-resolution microscopy has matured and moved from specialist optics labs into the hands of the biologist. Commercial microscope solutions exist for the three main variants for achieving optical super-resolution: single molecule localization microscopy (SMLM), stimulated emission depletion microscopy (STED), and structured illumination microscopy (SIM)[1,2]. Super-resolution microscopy via single molecule localization comes with an intrinsic trade-off: the spatial resolution attainable is dependent on accumulating a sufficient number of individual fluorophore localizations, limiting the temporal resolution. Imaging dynamic processes in live cells becomes problematic as one must adequately sample the movement of the structure of interest to prevent motion artifacts while acquiring enough localization events in that time to reconstruct an image. In order to meet these requirements, live cell SMLM demonstrations have obtained the required increase in fluorophore photoswitching rates by greatly increasing the excitation power, and this leads in turn to phototoxicity and oxidative stress, thereby limiting sample survival times and biological relevance[3]
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