A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

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.

Spatiotemporal analysis of host cell modification during herpes simplex virus 1 replication

“…Structured illumination images were collected on a custombuilt structured illumination microscopy (SIM) setup, modified from one which has been described before in detail [38]. A 60×/1.2NA water immersion lens (UPLSAPO60XW, Olympus, Tokyo, Japan) was used to focus the structured illumination pattern onto the sample and captured the samples' fluorescence emission light which was detected with a sCMOS camera (C11440, Hamamatsu, Hamamatsu-City, Japan).…”

Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction

“…In each microscope setup, the illumination patterns were produced by a high-speed ferroelectric liquid crystal on silicon (FLCOS) microdisplay (SXGA-3DM, Forth Dimension Displays, 13.6 µm pixel pitch). This particular FLCOS microdisplay has been used previously in SIM [14,16,25,29,30,36,37,[45][46][47][48], and in other optical sectioning systems such as programmable array microscopy (PAM) [38,42,49]. The display was illuminated by a home-built, three channel LED system based on high power LEDs (PT-54 or PT-120 with DK-114N or DK-136M controller, Luminous Devices) with emission maxima at 460 nm, 525 nm, and 623 nm.…”

Simultaneous multicolour imaging using quantum dot structured illumination microscopy

“…In the past decades, various SR techniques have been developed, including stimulated emission depletion (Hell & Wichmann, 1994), photoactivated localization microscopy (Betzig et al, 2006), stochastic optical reconstruction microscopy (Rust et al, 2006), structured illumination microscopy (SIM) (Gustafsson, 2000; (Dertinger et al, 2009). However, to our knowledge, most of the multicolour SIM schemes (Fiolka et al, 2012;Brunstein et al, 2013;Li et al, 2015;Young et al, 2016) acquire image of each colour channel sequentially. In addition, SIM is compatible with fluorescent markers used in conventional fluorescence microscopy.…”

“…Therefore, multicolour SIM is a handy tool used by biologists for the analysis of subcellular structures with high specificity, thus providing information of multicolour colocalization and dynamic interactions between organelles (Brunstein et al, 2013;Li et al, 2015). However, to our knowledge, most of the multicolour SIM schemes (Fiolka et al, 2012;Brunstein et al, 2013;Li et al, 2015;Young et al, 2016) acquire image of each colour channel sequentially. Therefore, the imaging speed of these multicolour SIM schemes is at least N times (assuming N colour channels) slower than monochromatic imaging, thus constraining the investigation of real-time interactions among subcellular structures.…”