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Stepp and colleagues present hybrid-EDA, an event-driven acquisition (EDA) that enables gentle investigation of rare mitochondrial events. This approach combines continuous, low-phototoxicity phase-contrast surveillance with event-triggered fluorescence imaging, powered by dynamics-aware machine-learning event detection.

In this Viewpoint, experts discuss resolution and common trade-offs in super-resolution microscopy, aiming to improve how biologists use the technology.

Super-resolution microscopy is poised to revolutionize our understanding of the workings of the cell. But the technology still has some limitations, and these must be taken into consideration if widespread application is to yield biological insight.

Imaging technologies drive discovery in cell biology. Innovations in microscopy hardware, imaging methods and computational analysis of large-scale, complex datasets can increase imaging resolution, definition and allow access to new biology. We asked experts at the leading edge of biological imaging what they are most excited about when it comes to microscopy in cell biology and what challenges need to be overcome to reach these goals.

Whether bacterial replisome progression follows a “factory” or “track” model remains a long-standing controversy. Here, the authors find that cells can switch between the two models, in a process governed by dynamic chromosome organization processes.

Multifocal flat illumination for field-independent imaging (mfFIFI) enables patterned illumination over an extended field of view. Integration with instant structured illumination microscope allowed for high-speed, multicolor, volumetric super-resolution imaging over 100 × 100 µm².

Mitochondrial fission at the organelle periphery generates small daughter mitochondria that are removed by mitophagy whereas fission at the midzone leads to proliferation.

Super-resolution microscopy at ångström precision could pave the way to optical structural biology in cells.

TIRF imaging is limited by the size and uniformity of the illumination. Here the authors present a waveguide solution to create a large area of uniform evanescent illumination suitable for single molecule imaging coupled with a customised sample holder containing a reservoir for DNA-PAINT solutions.

Event-driven acquisition uses neural-network-based recognition of specific biological events to trigger switching between slow and fast super-resolution imaging, enriching the capture of interesting events with high spatiotemporal resolution.

Spotiflow uses deep learning for subpixel-accurate spot detection in diverse 2D and 3D images. The improved accuracy offered by Spotiflow enables improved biological insights in both iST and live imaging experiments.

An epi-illumination system based on microlens arrays enables field-independent imaging of multiple cells with nanoscale resolution and large field of views.

Phase separation concentrates mitochondrial RNA granules. Here Rey et al., show that mitochondrial RNA granules (MRGs) behaviour is consistent with liquid–liquid phase separation (LLPS) and their fusion coincides with mitochondrial remodelling.

A computational and analytical framework enables multicolor 3D particle reconstruction of protein complexes from 2D images. The authors demonstrate the power of the approach by reconstructing native proteins within the human centriole.

Oncogenic gain-of-function EZH2 mutations alter gene–promoter interactions and lead to silencing 1,034 of genes within topologically associating domains, thus resulting in inactivation of multiple tumor-suppressor genes.

Actin condenses at the lamellipodium of migrating cells to form arc-like bundles parallel to the leading edge. During the retraction phase of the edge movement, these arcs are shown to be displaced towards the rear of the lamella, and their movement slows down when they join focal adhesions. Actin arcs thus provide a spatiotemporal connection between the lamellipodium and the lamella.

Improved photoactivatable red fluorescent proteins are generated by including properties desirable for photoactivated localization microscopy (PALM) as selection criteria. The PAmCherry proteins are superior tags for one- and two-color PALM in fixed cells, among other applications. Also in this issue, McKinney et al. present an improved version of the green-to-red EosFP protein.

Fluorescent probes for bioimaging need to exhibit bright fluorescence, be biocompatible and offer several alternatives for attachment to biomolecules of interest. Here, a near-infrared silicon–rhodamine fluorophore is introduced that can be coupled to intracellular proteins in live cells and tissues and can be exploited for super-resolution microscopy.

The prevailing challenge in live-cell fluorescence microscopy is capturing intra-cellular dynamics while preserving cell viability. Alongside developments of microscopy hardware, computational methods — especially those based on machine learning — are powerful tools to improve the signal-to-noise ratio, spatial resolution, temporal resolution and multi-colour capacity of live-cell imaging.

This Primer explains the central concepts of single-molecule localization microscopy (SMLM) before discussing experimental considerations regarding fluorophores, optics and data acquisition, processing and analysis. The Primer further describes recent high-impact discoveries made by SMLM techniques and concludes by discussing emerging methodologies.

In budding yeast, glucose withdrawal, via the Rag GTPases, leads to TORC1 inhibition through its re-organization into a giant, vacuole-associated helix named a TOROID (TORC1 organized in inhibited domain).

This Analysis reports a comparison of current software packages for single-molecule localization in localization-based super-resolution imaging. Performance of the participating software on synthetic, biologically inspired ground-truth data was assessed by multiple criteria.