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Synaptic plasticity ensures functionality during perturbations and enables memory formation. Here, the authors describe homeostatic functional and nano-modular active zone modifications for immediate and long-lasting enhancement of neurotransmitter release, and identify Unc13 as a presynaptic molecular target for homeostatic potentiation and learning.

Existing DNA stains for live cell microscopy are either toxic, require illumination with blue light, or are not compatible with super-resolution microscopy. Here the authors develop SiRHoechst, a non-toxic far-red DNA stain that is compatible with super-resolution microscopy.

STED nanoscopy enables sub-diffraction imaging with a wide range of fluorescent probes. Here, the authors show that a bright and very photostable class of fluorescent quantum dots can be super-resolved with STED as biolabels in cellular contexts.

A stimulated-emission-depletion-based fluorescence localization and super-resolution microscopy concept that is capable of attaining a spatial resolution down at the size scale of the fluorophores themselves and a localization precision of 1–3 nm in standard deviation is reported.

Quantum gates based on geometric phase shifts offer a promising approach for the realization of fault-tolerant quantum computing. Using nitrogen-vacancy centre qubits in diamond, this study experimentally realises a high-fidelty, non-adiabatic, non-Abelian holonomic single-qubit gate at room temperature.

The central maximum of a diffraction pattern is commonly used to resolve point-like scatterers. Now spatial resolution far below the diffraction limit is achieved by using the scattering minima.

Sub-nanometer localization precision is achieved with all-optical microscopy.

A dynamic model of the 4Pi point spread function enables localization microscopy with exceptional three-dimensional resolution and a simpler optical design. 4Pi-STORM images of neurons and mitochondria reveal new details of nanoscale protein and nucleic acid organization.

Minimal photon fluxes (MINFLUX) has enabled molecule-scale resolution in fluorescence microscopy but this had not been shown in standard, broadly applicable microscopy platforms. Here the authors report a solution to allow normal fluorescence microscopy while also providing 1-3 nm 3D resolution.

Activation of caged fluorophores has mostly relied on the absorption of either a single ultraviolet photon or two NIR photons. Here, the authors show that two green photons (515 nm) can substitute for a single photon ( ~ 260 nm) to activate rhodamine-based dyes, thus enabling STED imaging in thick samples.

The design of photoactivatable fluorophores—which are required for some super-resolution fluorescence microscopy methods—usually relies on light-sensitive protecting groups imparting lipophilicity and generating reactive by-products. Now, it has been shown that by exploiting a unique intramolecular photocyclization, bright and highly photostable fluorophores can be rapidly generated in situ from appropriately substituted 1-alkenyl-3,6-diaminoxanthone precursors.

Brakemann et al. present a reversibly photoswitchable fluorescent protein, called Dreiklang, that can be turned on and off at wavelengths distinct from those used for imaging. They show that the protein is advantageous for studying protein dynamics in living cells and for super-resolution imaging.

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.

The ability to discriminate objects along the optic axis is a benchmark for three-dimensional imaging techniques. Here, the authors combine metastable-state switching and opposing objective lenses to suppress out-of-focus background and record three-dimensional nanoscale images of living cells.

Advances in MINFLUX nanoscopy enable multicolor imaging over large fields of view, bringing true nanometer-scale fluorescence imaging to labeled structures in fixed and living cells.

Super-resolution microscopy using wavelengths in the near infrared (NIR) optical window is particularly appealing for live cell and tissue imaging, yet largely unexplored. Here the authors present NIR-STED nanoscopy of living mammalian cells using the new bacteriophytochrome-based fluorescent protein SNIFP.

Clustering of proteins in the plasma membrane plays an important role in the regulation of both cellular signalling and membrane remodelling. Milovanovic et al.demonstrate that mismatch between transmembrane domain length and the lipid bilayer thickness is sufficient to drive clustering of SNARE proteins.

The function of voltage-gated calcium channels in T cells is not well understood and controversial. Here the authors report that a regulatory beta subunit of voltage-gated calcium channels regulates T cell function despite no evidence that these channels were functional within T cells during activation.

Fluorescence microscopy is the most popular way to image biomolecules, but it leaves many of them in the dark. Non-fluorescent, light-absorbing molecules can now be viewed by a method that turns them into mini-lasers.

By exploiting a second off state of a reversibly switchable fluorophore, a general approach that can reduce photobleaching and enhance resolution of coordinate-targeted fluorescence nanoscopy has been demonstrated.

MINSTED quantifies tiny movements of individual biomolecules with high spatiotemporal precision to successfully resolve the steps of the molecular motor protein kinesin-1 labeled with a single fluorophore as it switches protofilaments.

Two incoherently superimposed orthogonal standing waves are used to create a pattern of 116,000 'doughnuts' for fast, highly parallelized coordinate-targeted super-resolution microscopy of living cells, with a large field of view.

Analyzing the first and higher-order moments of the diffraction spot of a 4Pi fluorescence detection scheme facilitates two-color, three-dimensional super-resolution microscopy with ~6 nm axial and ~8–23 nm lateral resolution in a layer ~650 nm thick.

To mark the 15th anniversary of Nature Methods, we asked scientists from across diverse fields of basic biology research for their views on the most exciting and essential methodological challenges that their communities are poised to tackle in the near future.

A fluorescence microscope relying entirely on focused light allows the generation of spherical focal fluorescence spots much smaller than the wavelength of light. This development, termed isoSTED, overcomes the resolution limitation imposed by the diffraction of light and permits three-dimensional nanoscale imaging inside cells with common fluorophores.

A simple yet powerful super-resolution imaging approach based on switching off ordinary fluorophores and localizing those remaining or regaining fluorescence is illustrated using continuous widefield illumination and imaging of fixed and living cells labeled with rhodamine-derived dyes or fluorescent proteins. Biteen et al., also in this issue, describe related work using the ordinary fluorophore of EYFP for super-resolution imaging.

Interactions between synaptotagmin-1 and the SNARE syntaxin-1 are known to mediate synaptic-vesicle exocytosis. Fusion experiments with artificial lipid membranes combined with the crystal structure of synaptotagmin's C2B domain bound to phosphoserine indicate that PIP2 clusters, organized by syntaxin, act as molecular beacons for vesicle docking and direct Ca²⁺-dependent membrane fusion.

The performance of low-power, continuous-wave stimulated emission depletion microscopy is improved by combining pulsed excitation with time-gated detection. This combination also simplifies super-resolution fluorescence correlation spectroscopy.

Single DNA-binding proteins can be tracked on densely covered DNA at high spatial and temporal resolution and in the presence of high protein concentrations by using a technique that combines optical tweezers, confocal fluorescence microscopy and stimulated emission depletion (STED) nanoscopy.

Mapping the distribution of fluorescence molecules, rather than just their emission intensity, can improve super-resolution fluorescence microscopy. Here, the authors present a general solution for rendering the number of fluorescent molecules recorded by confocal or STED microscopy.

Examining real-world data that tested different headlines for the same news story on real news readers, Robertson et al. find that people are more likely to click on a headline when it contains negative words compared to positive words.

A systematic exploration of MINFLUX nanoscopy with DNA-PAINT labeling leads to improved nanoscopy in fixed cells and MINFLUX imaging with increased multiplexing, as exemplified by three-color imaging of mitochondria in mammalian cells.

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.

Here, subdiffraction-resolution STED fluorescence microscopy is used to detect the diffusion of single lipids or GPI-anchored proteins on the plasma membrane of a living cell. Tuning the probing spot area ∼70-fold below that of a confocal microscope reveals that unlike phosphoglycerolipids, sphingolipids and GPI-anchored proteins are trapped for ∼10 ms in cholesterol-mediated complexes within <20 nm space.

Based on a far-field fluorescence-based optical super-resolution scheme – stimulated emission depletion microscopy – scientists resolve densely packed individual fluorescent colour centres inside crystals with a far-field spatial resolution of 5.8 nm without photobleaching. The approach will support future studies of solid-state single-photon sources and quantum optics.

Methods are reported for the combination of fluorescence nanoscopy using either stimulated emission depletion microscopy (STED) or photoactivated localization microscopy (PALM) with electron microscopy, to achieve correlative imaging in which the super-resolved fluorescence signal is placed in the context of cellular ultrastructure.

Synaptic assembly depends on trans-synaptic Neurexin/Neuroligin signalling. Here, Muhammad et al. show that Spinophilin, a pre-synaptic scaffolding protein, interacts with Neurexin, in competition with Syd-1, to regulate the formation and function of synaptic active zones at Drosophilaneuromuscular junctions.

Alborzinia et al. report that MYCN-amplified neuroblastoma undergoes ferroptosis in the absence of intracellular cysteine, suggesting a combination of cysteine depletion and concomitant GPX4 inactivation as a potential therapeutic approach.

Using super-resolution microscopy and cryo-electron microscopy, Stoldt et al. show that mitochondrial transcript translation and OXPHOS complex assembly are spatially partitioned within the mitochondrial membrane.