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Flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) in the brain is important for clearance of metabolic waste. Arterial pulsations are thought to drive flow, but this has never been quantitatively shown. We used particle tracking to quantify CSF flow velocities in PVSs of live mice. CSF flow is pulsatile and driven primarily by the cardiac cycle. The speed of the arterial wall matches that of the CSF, suggesting arterial wall motion is the principal driving mechanism, via a process known as perivascular pumping. Increasing blood pressure leaves the artery diameter unchanged but changes the pulsations of the arterial wall, increasing backflow and thereby reducing net flow in the PVS. Perfusion-fixation alters the normal flow direction and causes a 10-fold reduction in PVS size. We conclude that particle tracking velocimetry enables the study of CSF flow in unprecedented detail and that studying the PVS in vivo avoids fixation artifacts. Arterial pulsations are thought to drive CSF flow through perivascular spaces (PVSs), but this has never been quantitatively shown. Using particle tracking to quantify CSF flow velocities in PVSs of live mice, the authors show that flow speeds match the instantaneous speeds of the pulsing artery walls that form the inner boundaries of the PVSs.

Temporal focusing, with its ability to focus light in time, enables scanless illumination of large surface areas at the sample with micrometer axial confinement and robust propagation through scattering tissue. In conventional two-photon microscopy, widely used for the investigation of intact tissue in live animals, images are formed by point scanning of a spatially focused pulsed laser beam, resulting in limited temporal resolution of the excitation. Replacing point scanning with temporally focused widefield illumination removes this limitation and represents an important milestone in two-photon microscopy. Temporal focusing uses a diffusive or dispersive optical element placed in a plane conjugate to the objective focal plane to generate position-dependent temporal pulse broadening that enables axially confined multiphoton absorption, without the need for tight spatial focusing. Many techniques have benefitted from temporal focusing, including scanless imaging, super-resolution imaging, photolithography, uncaging of caged neurotransmitters and control of neuronal activity via optogenetics. This Review discusses temporal focusing microscopy and its applications in neuroscience for imaging and optogenetic activation.

Simultaneous imaging of neural activity in large regions of the mouse brain at subcellular resolution is made possible with a wide field-of-view two-photon microscope. Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to ∼1 mm 2 , precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm 2 ) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing.

Simultaneous multiview light-sheet microscopy using two illumination and two detection arms with one- or two-photon illumination is coupled to a fast data acquisition framework and analysis pipeline for quantitative imaging and tracking of individual cells and the developing nervous system throughout a living fly embryo. A related paper by Krzic et al . is also in this issue. Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo . SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.

Imaging the activity of neurons that are widely distributed across brain regions deep in scattering tissue at high speed remains challenging. Here, we introduce an open-source system with Dual Independent Enhanced Scan Engines for Large field-of-view Two-Photon imaging (Diesel2p). Combining optical design, adaptive optics, and temporal multiplexing, the system offers subcellular resolution over a large field-of-view of ~25 mm 2 , encompassing distances up to 7 mm, with independent scan engines. We demonstrate the flexibility and various use cases of this system for calcium imaging of neurons in the living brain. Imaging of neuronal activity across distant brain regions is challenging. Here, the authors introduce a two-photon microscope with two independently controlled scan engines, and demonstrate calcium imaging with subcellular resolution in brain regions up to 7 mm apart simultaneously.

Scattering in biological tissues is a major barrier for in vivo optical imaging of all but the most superficial structures. Progress toward overcoming the distortions caused by scattering in turbid media has been made by shaping the excitation wavefront to redirect power into a single point in the imaging plane. However, fast, non-invasive determination of the required wavefront compensation remains challenging. Here, we introduce a quickly converging algorithm for non-invasive scattering compensation, termed DASH, in which holographic phase stepping interferometry enables new phase information to be updated after each measurement. This leads to rapid improvement of the wavefront correction, forming a focus after just one measurement iteration and achieving an order of magnitude higher signal enhancement at this stage than the previous state-of-the-art. Using DASH, we demonstrate two-photon fluorescence imaging of microglia cells in highly turbid mouse hippocampal tissue down to a depth of 530  μ m. Wavefront shaping is used to overcome scattering in biological tissues during imaging, but determining the compensation is slow. Here, the authors use holographic phase stepping interferometry, where new phase information is updated after each measurement, enabling fast improvement of the wavefront correction.

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow. Precapillary sphincters are mural cells encircling an indentation of blood vessels where capillaries branch off from penetrating arterioles (PAs), but their existence and role in the brain is not fully understood. Here authors describe these structures at PAs in the cortex and show that they constrict during cortical spreading depolarization in mice.

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 μm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

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. Two-photon laser scanning microscopy of calcium dynamics using fluorescent indicators is a widely used imaging method for large-scale recording of neural activity in vivo . Here, we introduce volumetric two-photon imaging of neurons using stereoscopy (vTwINS), a volumetric calcium imaging method that uses an elongated, V-shaped point spread function to image a 3D brain volume. Single neurons project to spatially displaced 'image pairs' in the resulting 2D image, and the separation distance between projections is proportional to depth in the volume. To demix the fluorescence time series of individual neurons, we introduce a modified orthogonal matching pursuit algorithm that also infers source locations within the 3D volume. We illustrated vTwINS by imaging neural population activity in the mouse primary visual cortex and hippocampus. Our results demonstrated that vTwINS provides an effective method for volumetric two-photon calcium imaging that increases the number of neurons recorded while maintaining a high frame rate.

Adaptive optics and two-photon instant structured illumination microscopy are combined to provide improved super-resolution imaging within optically aberrating biological samples. We improve multiphoton structured illumination microscopy using a nonlinear guide star to determine optical aberrations and a deformable mirror to correct them. We demonstrate our method on bead phantoms, cells in collagen gels, nematode larvae and embryos, Drosophila brain, and zebrafish embryos. Peak intensity is increased (up to 40-fold) and resolution recovered (up to 176 ± 10 nm laterally, 729 ± 39 nm axially) at depths ∼250 μm from the coverslip surface.