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This study combines lattice light sheet microscopy and single molecule imaging to study protein dynamics and chromatin structure in live cells. The authors describe how nucleosomes and proteins move and are organised in relation to chromatin density.

ALI is an approach for analyzing voltage imaging data that is inspired by algorithms used in super-resolution microscopy. It allows resolving the activity of single neurons in densely labeled populations in scattering conditions.

3D ATAC-PALM integrates ATAC with super-resolution imaging for nanoscale views of the accessible genome. When combined with FISH, protein fluorescence and genetic perturbation, the method enables investigation of accessible chromatin in situ.

Neural mechanisms mediating information flow and processing in dendrites are not fully understood. Here the authors developed techniques to map bioelectrical excitations in the dendrites of neurons in acute slices of mouse brain tissue. They developed a holistic picture of the roles of dendritic excitations in spike back-propagation.

Combining the self-labeling HaloTag protein with synthetic environmentally sensitive fluorophores provides a platform for the construction of bright, far-red fluorescent calcium and voltage sensors with tunable photophysical and chemical properties.

Existing pH-sensitive red fluorescent protein probes don’t perform well in monitoring exocytosis and endocytosis. Here, the authors combine organic dyes with self-labeling tags or antibodies to develop semisynthetic protein conjugates that can image synaptic vesicle fusion events in living cells.

Genetically encoded voltage indicators (GEVIs) allow visualisation of fast action potentials in neurons but most are bright at rest and dimmer during an action potential. Here, the authors engineer electrochromic FRET GEVIs with fast, bright and positive-going fluorescence signals for in vivo imaging.

Reporter gene expression history in cells is recorded with engineered fluorescent protein fibers.

Live-cell single-molecule imaging of pioneer transcription factors revealed a ‘confined target search’ mechanism where they alternate between fast free diffusion in the nucleus and slower confined diffusion within compacted chromatin domains, leading to efficient pioneering on closed targets.

WHaloCaMP is a chemigenetic calcium indicator that can be combined with different rhodamine dyes for multiplexed or FLIM imaging in vivo, as demonstrated for calcium imaging in neuronal cultures, brain slices, Drosophila, zebrafish larvae and the mouse brain.

Here authors visualize dynamics GAGA pioneer factor (GAF) association with chromatin in vivo in Drosophila hemocytes. The characterization of GAF kinetics provides a temporal mechanism for maintenance of open chromatin for transcriptional responses to homeostatic, environmental and developmental signals.

Optopharmacological manipulation with ‘caged’ glutamate and GABA has enabled the study of these ligands’ cognate receptors, but other ligands such as tertiary amine drugs have not been amenable to caging. A new strategy yields a photoactivatable nicotine, PA-Nic, which allows manipulation of nicotinic acetylcholine receptors.

A general tuning strategy is introduced for improving the utility of rhodamines for biological imaging applications. The strategy yielded bright, versatile and bioavailable far-red and near-infrared ‘Janelia Fluor’ dyes.

Researchers developed DELTA, a method for brain-wide measurement of synaptic protein turnover with single-synapse resolution, providing a powerful tool to localize and study mechanisms underlying synaptic plasticity and learning.

Kim and coworkers describe a technique, EPSILON, to map AMPA receptor exocytosis, a proxy for synaptic plasticity, in mice. The authors demonstrated a correlation between AMPA receptor exocytosis and cFos expression during a fear conditioning experiment.

The molecular and cellular architecture of the organs in a whole mouse is revealed through optical clearing.

Courtney et al. found that the C-terminal amphipathic helix of a presynaptic protein, complexin, can dramatically remodel phospholipid bilayers. This activity enables complexin to increase the size and stability of SNARE-mediated fusion pores.

4Pi single-molecule switching microscopy combined with ‘salvaged fluorescence’ enables improved ratiometric imaging that bypasses chromatic aberrations and allows for multicolor whole-cell imaging with sub-10-nm localization precision.

A simple and general chemical structure change to a panel of cell-permeable small-molecule fluorophores increases their brightness and photostability, which will enable improved single-molecule studies and super-resolution imaging.

Lattice light-sheet and PAINT microscopy are combined to achieve low-background detection of dense molecular labels, yielding super-resolution localization microscopy images of intricate 3D structures within dividing cells and embryos.

Guidelines for fine-tuning spectral and chemical properties of fluorophore are introduced, resulting in four new Janelia Fluor dyes, JF₅₀₃, JF₅₂₅, JF₅₈₅ and JF₆₃₅, that span the visible region and align well with standard laser lines.

Cell-to-cell signaling networks, although poorly understood, guide tissue development, regulate tissue function and may become dysregulated in disease. Chemical biologists can develop the next generation of tools to untangle these complex and dynamic networks of interacting cells.

Photoactivatable derivatives of the bright and photostable Janelia Fluor dyes enable improved multicolor single-particle tracking and facile localization microscopy in cells.

This Perspective offers an overview of organic dyes commonly used as fluorescent labels and gives a chemist’s insight into their benefits and peculiarities.

Gerti Beliu et al. characterise the properties of tetrazine dyes, labelling proteins through site-specific introduction of a non-canonical amino acids followed by bioorthogonal click chemistry and evaluate the usefulness of tetrazine dyes for super-resolution microscopy.