Recent advances in tissue processing, labeling, and fluorescence microscopy are providing unprecedented views of the structure of cells and tissues at sub-diffraction resolutions and near single molecule sensitivity, driving discoveries in diverse fields of biology, including neuroscience. Biological tissue is organized over scales of nanometers to centimeters. Harnessing molecular imaging across intact, three-dimensional samples on this scale requires new types of microscopes with larger fields of view and working distance, as well as higher throughput. We present a new expansion-assisted selective plane illumination microscope (ExA-SPIM) with aberration-free 1.5 µm×1.5 µm×3 µm optical resolution over a large field of view (10.6×8.0 mm 2 ) and working distance (35 mm) at speeds up to 946 megavoxels/s. Combined with new tissue clearing and expansion methods, the microscope allows imaging centimeter-scale samples with 375 nm lateral and 750 nm axial resolution (4× expansion), including entire mouse brains, with high contrast and without sectioning. We illustrate ExA-SPIM by reconstructing individual neurons across the mouse brain, imaging cortico-spinal neurons in the macaque motor cortex, and visualizing axons in human white matter.

The gradual loss of cerebral white matter contributes to cognitive decline during aging. However, microvascular networks that support the metabolic demands of white matter remain poorly defined. We used in vivo deep multi-photon imaging to characterize microvascular networks that perfuse cortical layer 6 and corpus callosum, a highly studied region of white matter in the mouse brain. We show that these deep tissues are exclusively drained by sparse and wide-reaching venules, termed principal cortical venules, which mirror vascular architecture at the human cortical-U fiber interface. During aging, capillary networks draining into deep branches of principal cortical venules are selectively constricted, reduced in density, and diminished in pericyte numbers. This causes hypo-perfusion in deep tissues, and correlates with gliosis and demyelination, whereas superficial tissues become relatively hyper-perfused. Thus, age-related impairment of capillary-venular drainage is a key vascular deficit that contributes to the unique vulnerability of cerebral white matter during brain aging.

Progress in histological methods and in microscope technology has enabled dense staining and imaging of axons over large brain volumes, but tracing axons over such volumes requires new computational tools for 3D reconstruction of data acquired from serial sections. We have developed a computational pipeline for automated tracing and volume assembly of densely stained axons imaged over serial sections, which leverages machine learning-based segmentation to enable stitching and alignment with the axon traces themselves. We validated this segmentation-driven approach to volume assembly and alignment of individual axons over centimeter-scale serial sections and show the application of the output traces for analysis of local orientation and for proofreading over aligned volumes. The pipeline is scalable, and combined with recent advances in experimental approaches, should enable new studies of mesoscale connectivity and function over the whole human brain.

3-photon excitation laser-scanning microscopy enables fluorescence imaging deep in tissue beyond the range of 2-photon microscopy. Here, we report results of functional calcium imaging of neurons with 3-photon microscopy in non-human primates. We compare 2-photon and 3-photon excitation and characterize the performance of 3-photon microscopy at depths beyond the 2-photon limit.

Axonal connectomics entails tracing of dense axons over large brain volumes. Here, we describe a light-sheet imaging approach using scanned Bessel beam illumination and confocal slit detection and the results of simulation-guided optimization. We evaluate the performance of our approach for axon resolution and segmentation.

Expansion microscopy and light sheet imaging enable fine-scale resolution of intracellular features that comprise neural circuits. Most current techniques visualize sparsely distributed features across whole brains or densely distributed features within individual brain regions. Here, we visualize dense distributions of immunolabeled proteins across early visual cortical areas in adult macaque monkeys. This process may be combined with multiphoton or magnetic resonance imaging to produce multimodal atlases in large, gyrencephalic brains.

The motion/direction-sensitive and location-sensitive neurons are two major functional types in mouse visual thalamus that project to the primary visual cortex (V1). It has been proposed that the motion/direction-sensitive neurons mainly target the superficial layers in V1, in contrast to the location-sensitive neurons which mainly target the middle layers. Here, by imaging calcium activities of motion/direction-sensitive and location-sensitive axons in V1, we find no evidence for these cell-type specific laminar biases at population level. Furthermore, using a novel approach to reconstruct single-axon structures with identified in vivo response types, we show that, at single-axon level, the motion/direction-sensitive axons have middle layer preferences and project more densely to the middle layers than the location-sensitive axons. Overall, our results demonstrate that Motion/direction-sensitive thalamic neurons project extensively to the middle layers of V1, challenging the current view of the thalamocortical organizations in the mouse visual system.

We are applying light-sheet fluorescence microscopy with scanned Bessel beam illumination and confocal slit detection, combined with tissue expansion microscopy, to image dense axons over large brain volumes for axonal connectomics.

The organization and properties of the neural receptive fields in the visual cortex is a prominent factor in understanding the mechanism of neural coding. Here, we perform a systematic and standardized analysis of the structure, organization and properties of the receptive fields in the pan-excitatory neuronal population in the mouse primary visual cortex (area V1). We collected and analyzed a large-scale 2-photon volumetric imaging dataset from an 800x800x600 μm^3 volume in V1 of two mice. The visual responses from were recorded during presentation of a locally sparse noise stimulus to efficiently reconstruct the neural receptive fields. The locally sparse noise stimulus consisted of 9 degree black or white spots on a mean luminance gray background presented at ~3Hz. We designed and implemented a computational pipeline to identify responsive trials, ON and OFF subfields and their geometric properties. Using these experimental and computational pipelines, we found that OFF subfields tended to have larger areas than ON subfields, particularly in layer 2/3. On average, OFF subfields were 15.6 degree^2 larger than the ON subfields across all imaging depths. We also found that the average receptive field area decreased as a function of cortical depth. ON and OFF subfields in layer 5 were on average 22% (ON subfields) and 31.5% (OFF subfields) smaller than those in layer 2/3. Additionally, we found that the responsiveness rate to the sparse noise decreased in deeper layers of cortex. Neurons in layer 2/3 were about 4 times more likely to respond to our visual stimulus compared to neurons in layer 5. Our results provide a systematic and standardized survey of the receptive field properties in V1. The dataset and computational pipeline could serve as a valuable resource to the community to further study the visual responses of neurons in V1.

Motion/direction-sensitive and location-sensitive neurons are the two major functional types in mouse visual thalamus that project to the primary visual cortex (V1). It is under debate whether motion/direction-sensitive inputs preferentially target the superficial layers in V1, as opposed to the location-sensitive inputs, which preferentially target the middle layers. Here, by using calcium imaging to measure the activity of motion/direction-sensitive and location-sensitive axons in V1, we find evidence against these cell-type-specific laminar biases at the population level. Furthermore, using an approach to reconstruct axon arbors with identified in vivo response types, we show that, at the single-axon level, the motion/direction-sensitive axons project more densely to the middle layers than the location-sensitive axons. Overall, our results demonstrate that motion/direction-sensitive thalamic neurons project extensively to the middle layers of V1 at both the population and single-cell levels, providing further insight into the organization of thalamocortical projection in the mouse visual system.

AbstractTwo-photon fluorescence microscopy has been used extensively to probe the structure and functions of cells in living biological tissue. Two-photon excitation generates fluorescence from the focal plane, but also from outside the focal plane, with out-of-focus fluorescence increasing as the focus is pushed deeper into tissue. It has been postulated that the two-photon depth limit, beyond which results become inaccurate, is where in-focus and out-of-focus fluorescence are equal, which we term the balance depth. Calculations suggest that the balance depth should be at ∼600 µm in mouse cortex. Neither the two-photon depth limit nor the balance depth have been measured in brain tissue. We found the depth limit and balance depth of two-photon excitation in mice with GCaMP6 indicator expression in all layers of visual cortex, by comparing near-simultaneous two-photon and three-photon excitation. Two-photon and three-photon results from superficial locations were almost identical. two-photon results were inaccurate beyond the balance depth, consistent with the depth limit matching the balance depth for two-photon excitation. However, the two-photon depth limit and balance depth were at 450 µm, shallower than predicted by calculations. Our results were from tissue with a largely homogenous distribution of fluorophores. The expected balance depth is deeper in tissue with fewer fluorophores outside the focal plane and our results therefore establish a superficial bound on the two-photon depth limit in mouse visual cortex.

3-photon excitation enables in vivo fluorescence microscopy deep in densely labeled and highly scattering samples, while maintaining high resolution and contrast. We designed and characterized a dual-plane 3-photon microscope with temporal multiplexing and remote focusing, and performed simultaneous in vivo calcium imaging of two planes deep in the cortex of a transgenic mouse expressing GCaMP6s in nearly all excitatory neurons.

3-photon excitation enables in vivo fluorescence microscopy deep in densely labeled and highly scattering samples. To date, 3-photon excitation has been restricted to scanning a single focus, limiting the speed of volume acquisition. Here, for the first time to our knowledge, we implemented and characterized dual-plane 3-photon microscopy with temporal multiplexing and remote focusing, and performed simultaneous in vivo calcium imaging of two planes deep in the cortex of a pan-excitatory GCaMP6s transgenic mouse. This method is a straightforward and generalizable modification to single-focus 3PE systems, doubling the rate of volume (column) imaging with off-the-shelf components and minimal technical constraints.

Location-sensitive and motion-sensitive units are the two major functional types of feedforward projections from lateral genicular nucleus (LGN) to primary visual cortex (V1) in mouse. The distribution of these inputs in cortical depth remains under debate. By measuring the calcium activities of LGN axons in V1 of awake mice, we systematically mapped their functional and structural properties. Although both types distributed evenly across cortical depth, we found that they differ significantly across multiple modalities. Compared to the location-sensitive axons, which possessed confined spatial receptive fields, the motion-sensitive axons lacked spatial receptive fields, preferred lower temporal, higher spatial frequencies and had wider horizontal bouton spread. Furthermore, the motion-sensitive axons showed a strong depth-dependent motion direction bias while the location-sensitive axons showed a depth-independent OFF dominance. Overall, our results suggest a new model of receptive biases and laminar structure of thalamic inputs to V1. ![Figure][1] [1]: pending:yes

2-photon image quality degrades in volume-labeled tissue due to high background fluorescence. We apply 3-photon imaging to measuring functional responses in deep layers of primary visual cortex in pan-excitatory transgenic mice expressing GCaMP6s.

3-photon excitation permits fluorescence microscopy through the intact skull. We demonstrate 3-photon fluorescence imaging of neuronal structure and calcium activity in a chronic transcranial window preparation up to 300 μm into brain below the intact skull.

3-photon excitation permits fluorescence microscopy through the intact skull, but is highly sensitive to aberration. We estimate aberration encountered in focusing 1250 nm light through a glass-on-acrylic window for chronic transcranial in vivo imaging.

We combine light-field microscopy with scanning Bessel beam illumination to achieve 3D fluorescence imaging with uniformly high lateral resolution over 20 μm of axial thickness.

We apply deconvolution to light-field data acquired by light-field fluorescence microscopy with scanning Bessel-beam illumination to enhance resolution of 3D images. We demonstrate enhanced axial resolution and correction of aberration due to refractive index mismatch.