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Popularization of super-resolution imaging techniques has allowed cell biologists to probe cell structure and function in previously unattainable detail. These methodologies continue to evolve, with new improvements that allow tailoring the available techniques to a particular need and application. This collection showcases primary research articles, reviews and protocols and highlights these recent developments by exemplifying the new, interesting applications of super-resolution microscopy as well as related tool development. We hope that this compilation of works will inspire future research with the aim to resolve outstanding challenges and further expand the utility of super-resolution imaging across biological and medical disciplines.
A spatial resolution of 30 nm (=λ/31) exceeding the diffraction limit is achieved by super-resolution fluorescence microscopy. The nanoscopic imaging scheme can be applied to coherent quantum-mechanical systems such as quantum dots, as well as colour centres.
The ability to quantify the organization of cell membrane molecules is limited by the density of labeling and experimental conditions. Here, the authors use super-resolution optical fluctuation (SOFI) for molecular density and clustering analyses, and investigate nanoscale distribution of CD4 glycoprotein.
Studying interactions between lysosomes and mitochondria in living cells is difficult due to the limitations of existing probes. Here, the authors develop new cell-permeable fluorescent probes to image the dynamics of lysosomes and their physical interactions with mitochondria using super-resolution microscopy.
An automated system for data acquisition and analysis enables high-content screening localization microscopy and increases the throughput and information content of super-resolution microscopy methods such as dSTORM, DNA-PAINT and (spt)PALM.
Upconversion nanoparticles, which do not suffer from the photophysical artifacts that limit fluorescent molecules, offer an exciting opportunity for biological super-resolution imaging. Here, Zhan et al. develop an efficient STED mechanism using optimized lanthanide upconversion nanoparticles, enabling cytoskeleton nanoscopic imaging.
Ratiometric fluorescent pH probes are useful tools to monitor acidification of vesicles during endocytosis, but the size of vesicles is below the diffraction limit. Here the authors develop a family of ratiometric pH sensors for use in STED super-resolution microscopy, and optimize their delivery to endosomes.
Expansion microscopy, a technique for super-resolution imaging, is extended to clinical human tissue samples that are formalin fixed, paraffin embedded, stained and/or fresh frozen.
The measurement of molecular diffusion at sub-diffraction scales has been achieved in 2D space using STED-FCS, but an implementation for 3D diffusion is lacking. Here the authors present an analytical approach to probe diffusion in 3D space using STED-FCS and measure the diffusion of EGFP at different spatial scales.
In DNA-PAINT, transient binding of dye-labeled oligonucleotides to their target strands creates the ‘blinking’ required for stochastic nanoscopy. This protocol describes how to apply DNA-PAINT, from sample preparation to data processing.
Iterative expansion microscopy (iExM) is a strategy that achieves high resolution expansion microscopy by expanding samples multiple times. Expanding a sample twice enables ∼4.5 × 4.5 ∼20× physical expansion and ∼25 nm resolution.
This protocol describes the preparation of calibration bead slides, their use and additional strategies to reduce artifacts of structured illumination microscopy that will allow researchers to exploit the technique's full potential for biological applications.
This protocol describes how to prepare samples for labeling nuclei of cultured mammalian cells for 3D structured illumination microscopy of nuclear structures. Image acquisition, registration and downstream image analysis are also described.
Four different techniques for preparing and acquiring super-resolution CLEM data sets on aldehyde-fixed specimens are provided: Tokuyasu cryosectioning; whole-cell mount; cell unroofing and platinum replication; and resin embedding and sectioning.
New fluorescent biosensors enable the first super-resolution imaging of enzyme activity in live cells via fluorescence fluctuation increase by contact (FLINC).
Classical physics enabled subdiffraction-limited imaging has rarely been extended to the quantum regime. Here, Israelet al. develop a super-resolution localization microscopy based on non-classical photon statistics, enabling optical tracking of multiple quantum emitters.
Super-resolution optical microscopy based on stimulated emission depletion effects can now be performed at much lower light intensities than before by using bright upconversion emission from thulium-doped nanoparticles.
Stimulated emission double depletion addresses the issue of background in super-resolution imaging and quantitative microscopy through implementation of a two-pulse sequence in a modified stimulated emission depletion set-up. The measured background intensity is removed from each voxel in the acquired images thanks to time-resolved detection.
This protocol describes a detailed method for superresolution imaging of plant tissues by structured illumination microscopy (SIM). Details include microscope calibration, tissue preparation, image acquisition and evaluation of SIM images.