Collection |

Super-resolution microscopy

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. 

Reviews

This Perspective reviews nanoscopy via stimulated emission depletion (STED), focusing on challenges for biologists and how technical advances are helping to meet these challenges.

Perspective | | Nature Methods

An axon's function is dictated by its morphology and, thus, by the properties and organization of the axonal cytoskeleton. Leterrier et al. describe how advances in super-resolution and live-cell imaging are transforming our understanding of the molecular architecture of the axonal shaft.

Review Article | | Nature Reviews Neuroscience

Fluorescence nanoscopy enables the optical imaging of cellular components with resolutions at the nanometre scale. With the growing availability of super-resolution microscopes, nanoscopy methods are being increasingly applied. Quantitative, multicolour, live-cell nanoscopy and the corresponding labelling strategies are under continuous development.

Review Article | | Nature Reviews Molecular Cell Biology

Synaptic vesicles participate in neuronal communication by storing and releasing neurotransmitter molecules. The neurotransmitters can be detected using electrochemistry and mass spectrometry, and vesicle structural elements can be detected by super-resolution microscopy. This Review describes these analytical techniques and how they unravel the mechanisms of cell communication.

Review Article | | Nature Reviews Chemistry

In this Review, De Niz et al. discuss the contribution of key imaging tools to advances in our understanding of Plasmodium spp. biology and host–pathogen interactions over the past decade. These advances, pertaining to parasite structure and motility, as well as the liver and blood stages, have led to paradigm shifts in our knowledge of malaria.

Review Article | | Nature Reviews Microbiology

Optical and force nanoscopy enable visualization of intracellular and extracellular microbial structures with unprecedented resolution. This Review discusses the principles, advantages and limitations of the main optical and force nanoscopy techniques available.

Review Article | | Nature Microbiology

Applications in biology

The authors show that Munc13-1 molecules form multiple supramolecular self-assemblies that serve as vesicular release sites. Having multiple Munc13-1 assemblies affords a stable synaptic weight, which confers robustness of synaptic computation.

Article | | Nature Neuroscience

Bertocchi and colleagues describe the organization of cadherin-based adhesions using super-resolution microscopy. They find that α-catenin is important for vinculin localization and observe a conformational change in vinculin following its activation.

Article | | Nature Cell Biology

Methods & Protocols

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.

Article | open | | Nature Communications

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.

Article | open | | Nature Communications

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.

Article | open | | Nature Communications

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.

Article | open | | Nature Communications

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.

Article | open | | Nature Communications

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.

Protocol | | Nature Protocols

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.

Article | | Nature Methods

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.

Article | | Nature Photonics

News & Views

Spectacular images of the process of myosin II filament formation and organization in migrating cells are unveiled by super-resolution imaging. A combination of short- and long-range interactions with actin filaments is seen to play a critical role in filament partitioning and alignment into contractile actin arcs and stress fibres.

News & Views | | Nature Cell Biology

The ability to switch fluorophores on and off is key to performing super-resolution nanoscopy. To date, all switching schemes have been based on an incoherent response to the laser field. Now, a nanoscope that uses on–off coherent switching of quantum dots has been demonstrated.

News & Views | | Nature Photonics

Cadherin adhesion complexes have recently emerged as sensors of tissue tension that regulate key developmental processes. Super-resolution microscopy experiments now unravel the spatial organization of the interface between cadherins and the actin cytoskeleton and reveal how vinculin, a central component in cadherin mechanotransduction, is regulated by mechanical and biochemical signals.

News & Views | | Nature Cell Biology