Single-shot super-resolution total internal reflection fluorescence microscopy

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Abstract

We combine instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM), improving the lateral spatial resolution of TIRFM to 115 +/− 13 nm without compromising speed and enabling imaging frame rates up to 100 Hz over hundreds of time points. We apply instant TIRF-SIM to multiple live samples, achieving rapid, high contrast super-resolution imaging close to the coverslip surface.

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Most cited references 27

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A general method to improve fluorophores for live-cell and single-molecule microscopy

Jonathan Grimm, Brian English, Jiji Chen … (2015)

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here, we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

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ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.

Jian-Dong Li, Ye-Lin Shao, Bi-Chang Chen … (2015)

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

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Super-Resolution Video Microscopy of Live Cells by Structured Illumination

Peter Kner, Bryant B. Chhun, Eric Griffis … (2009)

Structured-illumination microscopy can double the resolution of the wide-field fluorescence microscope, but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed SIM that is capable of 100 nm resolution at frame rates up to 11 Hz for several hundred time frames. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila S2 cells in the total internal reflection (TIRF) mode.