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The GRIN lenses widely used for deep brain functional imaging suffer from a small measurement field of view due to strong fourth-order astigmatism. Here the authors report Geometric Transformation Adaptive Optics (GTAO) that corrects field-dependent astigmatism and enables large-volume in vivo imaging of deep mouse brain through 0.5 mm GRIN lenses.

Current systems for imaging calcium dynamics in the brains of non-human primates require the animal’s movement to be restricted. Here, the authors demonstrate a mesoscale calcium imaging device in a freely moving non-human primate which features a 20 mm² field of view.

A real-time analysis system was developed for an up to 500-megabyte-per-second image stream. This system can extract activities from up to 100,000 neurons in larval zebrafish brains and enables closed-loop perturbations of brain-wide neural dynamics at cellular resolution.

The fate of hematopoietic stem cells can be controlled by factors such as calcium ion concentration. Here the authors report an intravital ratiometric analysis method to measure extracellular calcium ion concentrations and absolute pH in mouse bone marrow.

The authors propose a new method for two-photon microscopy that can produce large cranial windows covering both the cerebrum and cerebellum of mice. The windows maintain transparency for over 5 months and allow multi-scale two-photon imaging of neuronal structures and functions in awake mice.

The authors develop an optical gearbox to accelerate the laser scanning microscopes, achieving flexible adjustment of imaging frame rate from tens of Hz to 1 kHz. The technology is validated through in vivo functional imaging of mice brains.

The formation and dissolution of ER-Mitochondria contacts is unclear. Here, authors show that the IP3 receptor traffics in and out of the contacts and, when trapped, improves calcium signaling to stimulate energy metabolism.

Fluorescence imaging of the spinal cord poses challenges, including depth of imaging. Here the authors describe a custom microscope and chronically implanted microprism that enables multicolor translaminar imaging of sensory and motor evoked activity in behaving mice, and show that spinal astrocytes show sensorimotor program-dependent calcium excitation.

In vivo calcium imaging at multiple depths simultaneously is shown using multifocal two-photon microscopy and spatiotemporal multiplexing. This technique involves scanning the sample with multiple beams in parallel at different axial planes and is applied to monitor neuronal network activity in multiple cortical layers of an anesthetized mouse.

This paper reports the use of light-field microscopy for fast, large-scale imaging of neuronal activity in vivo. It is applied to image the entire animal in the worm and the whole brain in zebrafish.

The image of a fluorescent object hidden behind an opaque layer can be retrieved non-invasively by exploiting the correlation properties of the speckle pattern produced by illuminating the object through the layer using laser light.

By targeting calcium indicators to primary cilia, micrometer-long protrusions from the cellular plasma membrane, the authors measure Ca²⁺ signaling in these sensory organelles.

Signals about head orientation and movement in the vestibular periphery are fundamental to the sense of balance and motion, but difficult to measure systematically during head motion. Here, the authors build a microscope that visualizes neural activity in hair cells and vestibular ganglion cells during 360° head tilt and vibration in zebrafish larvae, and reveal a topographic organization of direction- and static/dynamic stimulus-selective responses.

Functional brain imaging with two-photon microscopy is limited by a tradeoff between imaging area and acquisition speed. Here, the authors present Quadroscope, a flexible microscope which allows for simultaneous video rate acquisition of four independently targetable brain regions across 5 mm.

In vivo lensless imaging can be achieved via a phase mask that creates diffraction patterns with high-contrast contours, as shown for the imaging of calcium dynamics in the cortex of mice and of microvasculature in the oral mucosa of volunteers.

COMPACT allows imaging of large volumes in the brain through a periscope-like probe inside a glass capillary.

The FRET efficiency usually predominantly depends on the proximity of donor and acceptor. Here the authors report an anisotropy-based mode of FRET detection, FRET-induced Angular Displacement Evaluation via Dim donor (FADED), to allow quantification of the relative angle between donor and acceptor.

Light-field microscopy provides volumetric imaging at high speeds, but suffers from degradation in scattering tissue. Here, the authors present an incoherent multiscale scattering model which allows for quantitative 3D reconstruction in complex environments, and demonstrate dynamic imaging in vivo.

Two-photon microscopy across the fly brain using sensors that permit simultaneous measurement of neural activity and metabolic flux reveals global and local coordination of neural activity and energy metabolism.

Biosensors often report relative rather than absolute values. Here the authors report a method that utilises the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity: photochromism-enabled absolute quantification (PEAQ) biosensing.

SCAPE 2.0 is a versatile imaging platform that enables real-time three-dimensional microscopy of cellular function and dynamic motion in living organisms at over 100 volumes per second with minimal photodamage, and high-throughput structural imaging in fixed, cleared and expanded samples.

Although calcium signals are known to be critical for many cellular processes, how signaling elicits specific functions remains unclear. In visually striking work, Duan et al. reveal that networks of cytoplasmic nanocourses orchestrate cell activity by directing site-specific calcium signals.

Processing of two-photon calcium imaging data is generally time-consuming, especially for large fields of view. Here, the authors present CITE-On, a tool based on a convolutional neural network, enabling online automatic cell identification, segmentation, identity tracking, and trace extraction.

3D-CASH is a random-access microscopy approach that avoids in vivo motion artifacts by sampling each targeted neuron with a holographically shaped grid of illumination spots. The technology allows recording neuronal activity in the mouse cortex at a throughput of 20,000 neurons per second.

Currently, genetically encoded calcium indicators are not suitable for direct quantification. Here the authors engineer a fluorescence lifetime imaging calcium biosensor, Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS), and measure intracellular calcium concentrations in human-derived organoids.

Here, the authors present a two-photon light-sheet microscopy with an extended Bessel beam for a tunable field of view and reduced photodamage. They demonstrate long-term imaging of cellular dynamics over the whole body of medaka fish.

Three-photon microscopy in combination with adaptive optics-based aberration correction and ECG-triggered gating allows high-resolution imaging of neurons and astrocytes up to a depth of 1.4 mm in the mouse brain.

Current calcium-sensitive probes based on red fluorescent proteins are unsuitable for two-photon excitation at the near-infrared wavelengths commonly used for green fluorescent probes. Wu et al. use a structure-guided approach to engineer a red fluorescent probe with optimal two-photon excitation at these wavelengths.

A new imaging method enables deep imaging of neural networks.

Deep-Z uses deep learning to go from a two-dimensional snapshot to three-dimensional fluorescence images. The method improves imaging speed while reducing light dose, and was shown to be useful for accurate structural and functional imaging of neurons in Caenorhabditis elegans.

Zuend and colleagues show that an arousal-induced increase in cortical activity is accompanied by a surge in lactate in the extracellular space and a substantial lactate dip in astrocytes, followed by mobilization of lactate from glycogen stores and neuronal lactate increase.

Pulsatile GABA secretion from human beta cells via the volume regulatory anion channel (VRAC) and subsequent uptake by the GABA-permissive taurine transporter (TauT) is shown to regulate total insulin secretion and pulsatility.

High-density arrays of optical fibers enable monitoring and manipulation of neural activity at large scale across many brain regions. The multi-fiber arrays can be used in head-fixed tasks, in freely behaving animals and during social interactions.

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.

Recording the activity of neurons over large brain regions requires expanding the field of view of the optics without losing on spatial and temporal resolution. Here, the authors report a micro-opto-mechanical device that enables two-photon imaging across distant motor areas around 6 mm apart in the mouse.

Methods to directly label active neurons are still lacking. Here the authors develop CaMPARI2, a photoconvertible fluorescent protein sensor for neuronal activity with improved brightness and calcium binding kinetics, as well as an antibody to amplify the activated sensor signal in fixed samples.

Marmosets are an important model organism in neuroscience but there has only been limited success in training them on behavioral tasks. Here the authors report their ability to train marmosets in various motor tasks and simultaneously image neural dynamics in motor cortex with 2-photon imaging.

The use of intracellular calcium sensors provides important information about the dynamics of calcium signalling in cells. Here Suzuki et al. develop organelle-targeted sensors to simultaneously measure calcium concentrations in ER and mitochondria, and uncover novel insights into calcium flux in mitochondria.

Calcium-sensing fluorescent proteins such as TN-XXL are valuable tools for studying cellular function but, when expressed in mice, may affect animal physiology and behaviour. The authors of this paper create transgenic mice expressing TN-XXL and show that long-term expression of TN-XXL is tolerated well.

Two miniature microscopes with innovative light paths are described and applied to imaging of juvenile zebra finches and mice.

vTwINS enables high-speed volumetric calcium imaging via a V-shaped point spread function and a dedicated data-processing algorithm. Song et al. apply this strategy to image population activity in the mouse visual cortex and hippocampus.

FHIRM-TPM is a miniature two-photon microscope capable of imaging fluorescently labeled neurons in the brains of freely behaving mice. It allows for imaging of spines or recording of neural activity with a frame rate up to 40 Hz.

Quantitatively studying components of the presynapse requires high resolution optical methods. Here the authors use confocal microscopy as well as 2D- and 3D-STED nanoscopy to quantify the number and activity of active zone Ca²⁺ channels in inner hair cells.

Piezo ion channels transduce mechanical stimuli into biological signals but the underlying mechanism has remained elusive. Here, the authors use the selective agonist Yoda1 to identify molecular determinants of Piezo activation, providing mechanistic insights into Piezo-mediated mechanotransduction.

Two-photon scanning microscopy is inherently slow and thus limits volumetric calcium imaging. Prevedel et al. achieve increased volumetric imaging speed by tailoring the excitation volume via light sculpting.

Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

The authors use in vivo two-photon calcium imaging to examine the responses and network dynamics of layer 2/3 neurons in the primary auditory cortex of mice in response to pure tones. They find that local populations in A1 are heterogeneous, but, despite this, there was a higher than average noise correlation.

Using two-photon calcium imaging, the authors found that although tonotopy was present in mouse auditory cortex at a coarse scale, it was fractured on a fine scale. Intensity tuning appeared to have no topography at all, but subthreshold responses revealed that there was some clustering of neurons with similar response properties.

Unlike that of its main counterpart, the functional organization of the accessory olfactory bulb, important for detecting socially relevant odors, remains to be detailed. Here the authors map out Ca²⁺ signals from vomeronasal inputs to the accessory olfactory bulb in response to socially relevant compounds and find a non-chemotopic spatial organization.

This technique allows functional imaging of neurons in head-fixed Drosophila while the fly walks on an air-supported ball. Using a genetically encoded calcium sensor, the activity of motion-sensitive neurons in the fly optic lobe was recorded while the flies were presented with visual stimuli. Activity in these cells correlated with robust optomotor behavior in the walking flies.