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The fluorescent proteins mEos4a and mEos4b maintain their fluorescence and photoconversion after fixation with osmium. This property enables applications such as correlative super-resolution and electron microscopy.

A two-photon computed tomography approach, called scanned line angular projection microscopy, enables high-speed imaging at over 1 kHz frame rates, as demonstrated for glutamate imaging in the in vivo mouse brain.

Striatal cholinergic interneurons promote flexible behavior by unknown mechanisms. Here, authors show that spatially heterogeneous acetylcholine signals promote adaptive lose-shift choices in response to unexpected non-reward in a virtual Y-maze.

ATP has essential roles in cell signalling and energy homeostasis and biosensors to detect it have many potential applications. Here, the authors develop a new ATP sensor that can be targeted to the membrane or cytosol.

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.

GABA is the primary inhibitory neurotransmitter in the central nervous system. Using two-photon GABA imaging, Matsumoto et al. reveal over 40 GABA neuron types in the mouse retina, each uniquely filtering visual features.

An improved version of the green-to-red photoconverting EosPF is presented. mEos2 is a functional fusion partner for many cellular proteins and retains the high localization precision of EosFP in super-resolution imaging. Also in this issue, Subach et al. present an inducible mCherry variant for super-resolution imaging.

Microbial rhodopsins convert light into ion flux; in neurons, this can be used to control activity. New work shows that the opposite is also true: rhodopsins can be used to visualize neural activity.

'Spaghetti monster' fluorescent proteins combine the power of conventional fluorescent proteins with the benefits of commonly used epitopes. These probes are demonstrated to be extremely versatile in diverse imaging applications.

Cichon et al. show that ketamine induces a switch in the active population of excitatory neurons across cortical layers and regions that contributes to impairments in sensory processing characteristic of a dissociative-like state.

Folded proteins are composed of secondary structures, α-helices and β-sheets, that are generally assumed to be stable. Here, the authors combine computational prediction with experimental validation to show that many sequence-diverse NusG protein domains switch completely from α-helix to β-sheet folds.

The engineered hyperfolder YFP (hfYFP) and variants offer unprecedented chemical and thermal stability, making them versatile probes for microscopy as well as for challenging applications like correlative light and electron microscopy and expansion microscopy.

iGluSnFR variants with improved signal-to-noise ratios and targeting to postsynaptic sites have been developed, enabling the analysis of glutamatergic neurotransmission in vivo as illustrated in the mouse visual and somatosensory cortex.

Astrocyte calcium increases can alter brain state, but their dynamics during different behaviors have not been fully described. Here, the authors use multicore fiber optic imaging in freely moving mice to show that astrocyte engagement in behavior is influenced by the motivational state.

Using large-scale screening and structure-guided mutagenesis, fast and sensitive GCaMP sensors are developed and optimized with improved kinetics without compromising sensitivity or brightness.

Single-cell transcriptomics of more than 20,000 cells from two functionally distinct areas of the mouse neocortex identifies 133 transcriptomic types, and provides a foundation for understanding the diversity of cortical cell types.

Hippocampal place cells contribute to navigation and memory formation. Here, the authors use in vivo glutamate imaging to reveal patterns of excitatory input received by place cell dendrites and find more spatially tuned and functionally organized inputs arriving in the place field.

How neurotransmitter release relates to presynaptic calcium dynamics is not fully understood. Here the authors develop an approach based on FLIM and optical quantal analysis for simultaneous imaging of calcium dynamics and glutamate release at presynaptic boutons.

The ‘jGCaMP7’ sensors are four genetically encoded calcium indicators with better sensitivity than state-of-the-art GCaMP6 and specifically improved for applications such as neuropil or wide-field imaging. The sensors are validated in vivo in both flies and mice.

Light sensing outside the eyes is common in many animals but is usually confined to specialized organs. Here, the entire body wall of the fruitfly larva is found to be tiled with blue- and ultraviolet-light sensing neuronal dendrites, which are essential for the larva's innate light-avoidance behaviour. The phototransduction machinery used by these neurons is distinct from other Drosophila photoreceptor molecules but similar to a system recently identified in nematode neurons.

Sensitive protein sensors of calcium have been created; these new tools are shown to report neural activity in cultured neurons, flies and zebrafish and can detect single action potentials and synaptic activation in the mouse visual cortex in vivo.

Swapan Nath, Sang-Cheol Bae and colleagues report the results of a large-scale association study of systemic lupus erythematosus in individuals of Asian ancestry. They identify several new susceptibility loci and find enrichment for signals near genes implicated in B cell and T cell function.

Transcriptional profiling and axonal reconstructions identify two types of pyramidal tract neuron in the motor cortex: one type projects to thalamic regions and produces early and persistent preparatory activity, and the other type projects to motor centres in the medulla and produces motor commands.

An improved version of the GCaMP genetically encoded calcium indicator, called GCaMP3, has higher calcium affinity and increased baseline fluorescence, dynamic range and stability. GCaMP3 performs better than existing genetically encoded calcium indicators in several assays and organisms, including in vivo imaging of neuronal signaling in worms, flies and mice.

The genetically encoded GABA sensor iGABASnFR allows visualizing GABA signaling in vivo. Its application is demonstrated in mouse slices, in the awake mouse and in behaving zebrafish.

A genetic multicolor cell-labeling technique for Droshophila melanogaster, Drosophila Brainbow, is described and applied to the study of neural circuits. This method implements a variant of the mouse Brainbow strategy in combination with specific neuronal targeting using the Gal-4–upstream activating sequence system to select for epitope-tagged proteins detectable with immunofluorescence. Also in this issue, Hadjieconomou et al. develop a similar strategy, Flybow, to select for membrane-tethered fluorescent proteins.

A single-wavelength genetically encoded sensor of extracellular glutamate is reported. The sensor—iGluSnFR—is bright and photostable under both one- and two-photon illumination and is shown to work for in vivo imaging in worms, zebrafish and mice.

Using two-color two-photon calcium imaging, the authors identified transformations of representations across synaptically connected pairs of neurons along a visual pathway to the Drosophila central complex. Neural responses to stimuli in the ipsilateral field are modulated by stimuli in the contralateral field, an effect that depends on past stimulus history.

An open-source library of analytical tools for mapping large-scale patterns of brain activity using cluster computing finds structure in two-photon imaging data from mouse and whole-brain light-sheet functional imaging data from behaving larval zebrafish. Vladimirov et al., also in this issue, describes the light-sheet functional imaging system used here.

Variants of the genetically encoded sensor iGluSnFR extend the range of conditions under which glutamate neurotransmission can be visualized. In addition, chromatic variants of iGluSnFR improve compatibility with various illumination schemes.

jYCaMP1, a yellow variant of the calcium indicator jGCaMP7, enables fast multicolor two-photon imaging at excitation wavelengths above 1,000 nm for use with popular ytterbium-doped fiber and modelocked semiconductor lasers.

Imaging of activity in long-range axons is reported in mice performing tactile object-localization with their whiskers; the feedback projection from the motor cortex to the somatosensory cortex provides information to integrate whisker movement information and touch, which are key components of object identification.

Innate differences between male and female behaviours must be inscribed in their respective genomes, but how these encode distinct neuronal circuits remains largely unclear. Focusing on sex specific responses to the cVA pheromone in fruitflies, a chain of four successive neurons carrying olfactory signals down to motor centres has been identified, with all male to female anatomical differences lying downstream of a conserved sensory cell. The techniques developed should help others in the task of neuronal circuit mapping, which remains daunting even for the relatively simple fly brain.

Two-photon calcium imaging has previously only been useful for imaging ongoing neuronal activity in the superficial cortical layers in vivo. Here the authors describe technology that enables imaging of sensory-evoked neuronal activity in layer 5 of adult mouse somatosensory cortex.

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.

Dombeck and colleagues describe a method for two-photon calcium imaging using a genetically encoded indicator in the hippocampus of awake, behaving mice. This powerful approach permits the recording of multiple hippocampal place cells' activity with subcellular resolution as the mice run on a track in a virtual reality environment.

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.