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

Accepted version
Peer-reviewed

Repository DOI


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Authors

Young, Laurence J 
Ströhl, Florian 
Kaminski, Clemens F 

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.

Description

Keywords

Color, Microscopy, Fluorescence

Journal Title

Journal of Visualized Experiments

Conference Name

Journal ISSN

1940-087X
1940-087X

Volume Title

Publisher

MYJoVE Corporation
Sponsorship
Engineering and Physical Sciences Research Council (EP/H018301/1)
Medical Research Council (MR/K02292X/1)
Medical Research Council (MR/K015850/1)
Wellcome Trust (089703/Z/09/Z)
Engineering and Physical Sciences Research Council (EP/G037221/1)
This work was supported by grants from the Leverhulme Trust, the Engineering and Physical Sciences Research Council [EP/H018301/1, EP/G037221/1]; Alzheimer Research UK [ARUK-EG2012A-1]; Wellcome Trust [089703/Z/09/Z] and Medical Research Council [MR/K015850/1, MR/K02292X/1]. We thank K. O’Holleran for assistance with the design of the microscope, and L. Shao and R. Heintzmann for useful discussions and suggestions.