Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples.

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope.

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.

Whole-mouse clearing and imaging at the cellular level with vDISCO.

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

Whole-brain microscopy reveals distinct temporal and spatial efficacy of anti-Aß therapies.

Many efforts targeting amyloid-ß (Aß) plaques for the treatment of Alzheimer's Disease thus far have resulted in failures during clinical trials. Regional and temporal heterogeneity of efficacy and dependence on plaque maturity may have contributed to these disappointing outcomes. In this study, we mapped the regional and temporal specificity of various anti-Aß treatments through high-resolution light-sheet imaging of electrophoretically cleared brains. We assessed the effect on amyloid plaque formation and growth in Thy1-APP/PS1 mice subjected to ß-secretase inhibitors, polythiophenes, or anti-Aß antibodies. Each treatment showed unique spatiotemporal Aß clearance, with polythiophenes emerging as a potent anti-Aß compound. Furthermore, aligning with a spatial-transcriptomic atlas revealed transcripts that correlate with the efficacy of each Aß therapy. As observed in this study, there is a striking dependence of specific treatments on the location and maturity of Aß plaques. This may also contribute to the clinical trial failures of Aß-therapies, suggesting that combinatorial regimens may be significantly more effective in clearing amyloid deposition.

The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples.

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.

Multiscale optical and optoacoustic imaging of amyloid-ß deposits in mice.

Deposits of amyloid-ß (Aß) in the brains of rodents can be analysed by invasive intravital microscopy on a submillimetre scale, or via whole-brain images from modalities lacking the resolution or molecular specificity to accurately characterize Aß pathologies. Here we show that large-field multifocal illumination fluorescence microscopy and panoramic volumetric multispectral optoacoustic tomography can be combined to longitudinally assess Aß deposits in transgenic mouse models of Alzheimer's disease. We used fluorescent Aß-targeted probes (the luminescent conjugated oligothiophene HS-169 and the oxazine-derivative AOI987) to transcranially detect Aß deposits in the cortex of APP/PS1 and arcAß mice with single-plaque resolution (8 µm) and across the whole brain (including the hippocampus and the thalamus, which are inaccessible by conventional intravital microscopy) at sub-150 µm resolutions. Two-photon microscopy, light-sheet microscopy and immunohistochemistry of brain-tissue sections confirmed the specificity and regional distributions of the deposits. High-resolution multiscale optical and optoacoustic imaging of Aß deposits across the entire brain in rodents thus facilitates the in vivo study of Aß accumulation by brain region and by animal age and strain.

Isotropic imaging across spatial scales with axially swept light-sheet microscopy.

Light-sheet fluorescence microscopy is a rapidly growing technique that has gained tremendous popularity in the life sciences owing to its high-spatiotemporal resolution and gentle, non-phototoxic illumination. In this protocol, we provide detailed directions for the assembly and operation of a versatile light-sheet fluorescence microscopy variant, referred to as axially swept light-sheet microscopy (ASLM), that delivers an unparalleled combination of field of view, optical resolution and optical sectioning. To democratize ASLM, we provide an overview of its working principle and applications to biological imaging, as well as pragmatic tips for the assembly, alignment and control of its optical systems. Furthermore, we provide detailed part lists and schematics for several variants of ASLM that together can resolve molecular detail in chemically expanded samples, subcellular organization in living cells or the anatomical composition of chemically cleared intact organisms. We also provide software for instrument control and discuss how users can tune imaging parameters to accommodate diverse sample types. Thus, this protocol will serve not only as a guide for both introductory and advanced users adopting ASLM, but as a useful resource for any individual interested in deploying custom imaging technology. We expect that building an ASLM will take ~1-2 months, depending on the experience of the instrument builder and the version of the instrument.

Deep learning is widely applicable to phenotyping embryonic development and disease.

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.

3D Reconstruction of the Clarified Rat Hindbrain Choroid Plexus.

The choroid plexus (CP) acts as a regulated gate between blood and cerebrospinal fluid (CSF). Despite its simple histology (a monostratified cuboidal epithelium overlying a vascularized stroma), this organ has remarkably complex functions several of which involve local interaction with cells located around ventricle walls. Our knowledge of CP structural organization is mainly derived from resin casts, which capture the overall features but only allow reconstruction of the vascular pattern surface, unrelated to the overlying epithelium and only loosely related to ventricular location. Recently, CP single cell atlases are starting to emerge, providing insight on local heterogeneities and interactions. So far, however, few studies have described CP spatial organization at the mesoscale level, because of its fragile nature and deep location within the brain. Here, using an iDISCO-based clearing approach and light-sheet microscopy, we have reconstructed the normal rat hindbrain CP (hCP) macro- and microstructure, using markers for epithelium, arteries, microvasculature, and macrophages, and noted its association with 4th ventricle-related neurovascular structures. The hCP is organized in domains associated to a main vessel (fronds) which carry a variable number of villi; the latter are enclosed by epithelium and may be flat (leaf-like) or rolled up to variable extent. Arteries feeding the hCP emerge from the cerebellar surface, and branch into straight arterioles terminating as small capillary anastomotic networks, which run within a single villus and terminate attaching multiple times to a large tortuous capillary (LTC) which ends into a vein. Venous outflow mostly follows arterial pathways, except for the lateral horizontal segment (LHS) and the caudal sagittal segment. The structure of fronds and villi is related to the microvascular pattern at the hCP surface: when LTCs predominate, leaflike villi are more evident and bulge from the surface; different, corkscrew-like villi are observed in association to arterioles reaching close to the CP surface with spiraling capillaries surrounding them. Both leaf-like and corkscrew-like villi may reach the 4th ventricle floor, making contact points at their tip, where no gap is seen between CP epithelium and ependyma. Contacts usually involve several adjacent villi and may harbor epiplexus macrophages. At the junction between medial (MHS) and lateral (LHS) horizontal segment, arterial supply is connected to the temporal bone subarcuate fossa, and venous outflow drains to a ventral vein which exits through the cochlear nuclei at the Luschka foramen. These vascular connections stabilize the hCP overall structure within the 4th ventricle but make MHS-LHS joint particularly fragile and very easily damaged when removing the brain from the skull. Even in damaged samples, however, CP fronds (or isolated villi) often remain strongly attached to the dorsal cochlear nucleus (DCN) surface; in these fronds, contacts are still present and connecting "bridges" may be seen, suggesting the presence of real molecular contacts rather than mere appositions.

Tutorial: practical considerations for tissue clearing and imaging.

Tissue clearing has become a powerful technique for studying anatomy and morphology at scales ranging from entire organisms to subcellular features. With the recent proliferation of tissue-clearing methods and imaging options, it can be challenging to determine the best clearing protocol for a particular tissue and experimental question. The fact that so many clearing protocols exist suggests there is no one-size-fits-all approach to tissue clearing and imaging. Even in cases where a basic level of clearing has been achieved, there are many factors to consider, including signal retention, staining (labeling), uniformity of transparency, image acquisition and analysis. Despite reviews citing features of clearing protocols, it is often unknown a priori whether a protocol will work for a given experiment, and thus some optimization is required by the end user. In addition, the capabilities of available imaging setups often dictate how the sample needs to be prepared. After imaging, careful evaluation of volumetric image data is required for each combination of clearing protocol, tissue type, biological marker, imaging modality and biological question. Rather than providing a direct comparison of the many clearing methods and applications available, in this tutorial we address common pitfalls and provide guidelines for designing, optimizing and imaging in a successful tissue-clearing experiment with a focus on light-sheet fluorescence microscopy (LSFM).

Output-Specific Adaptation of Habenula-Midbrain Excitatory Synapses During Cocaine Withdrawal.

Projections from the lateral habenula (LHb) control ventral tegmental area (VTA) neuronal populations' activity and both nuclei shape the pathological behaviors emerging during cocaine withdrawal. However, it is unknown whether cocaine withdrawal modulates LHb neurotransmission onto subsets of VTA neurons that are part of distinct neuronal circuits. Here we show that, in mice, cocaine withdrawal, drives discrete and opposing synaptic adaptations at LHb inputs onto VTA neurons defined by their output synaptic connectivity. LHb axons innervate the medial aspect of VTA, release glutamate and synapse on to dopamine and non-dopamine neuronal populations. VTA neurons receiving LHb inputs project their axons to medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and lateral hypothalamus (LH). While cocaine withdrawal increases glutamate release from LHb onto VTA-mPFC projectors, it reduces presynaptic release onto VTA-NAc projectors, leaving LHb synapses onto VTA-to-LH unaffected. Altogether, cocaine withdrawal promotes distinct adaptations at identified LHb-to-VTA circuits, which provide a framework for understanding the circuit basis of the negative states emerging during abstinence of drug intake.

Sensory and Behavioral Components of Neocortical Signal Flow in Discrimination Tasks with Short-Term Memory.

In the neocortex, each sensory modality engages distinct sensory areas that route information to association areas. Where signal flow converges for maintaining information in short-term memory and how behavior may influence signal routing remain open questions. Using wide-field calcium imaging, we compared cortex-wide neuronal activity in layer 2/3 for mice trained in auditory and tactile tasks with delayed response. In both tasks, mice were either active or passive during stimulus presentation, moving their body or sitting quietly. Irrespective of behavioral strategy, auditory and tactile stimulation activated distinct subdivisions of the posterior parietal cortex, anterior area A and rostrolateral area RL, which held stimulus-related information necessary for the respective tasks. In the delay period, in contrast, behavioral strategy rather than sensory modality determined short-term memory location, with activity converging frontomedially in active trials and posterolaterally in passive trials. Our results suggest behavior-dependent routing of sensory-driven cortical signals flow from modality-specific posterior parietal cortex (PPC) subdivisions to higher association areas.

Developmental divergence of sensory stimulus representation in cortical interneurons.

Vasocative-intestinal-peptide (VIP+) and somatostatin (SST+) interneurons are involved in modulating barrel cortex activity and perception during active whisking. Here we identify a developmental transition point of structural and functional rearrangements onto these interneurons around the start of active sensation at P14. Using in vivo two-photon Ca2+ imaging, we find that before P14, both interneuron types respond stronger to a multi-whisker stimulus, whereas after P14 their responses diverge, with VIP+ cells losing their multi-whisker preference and SST+ neurons enhancing theirs. Additionally, we find that Ca2+ signaling dynamics increase in precision as the cells and network mature. Rabies virus tracings followed by tissue clearing, as well as photostimulation-coupled electrophysiology reveal that SST+ cells receive higher cross-barrel inputs compared to VIP+ neurons at both time points. In addition, whereas prior to P14 both cell types receive direct input from the sensory thalamus, after P14 VIP+ cells show reduced inputs and SST+ cells largely shift to motor-related thalamic nuclei.

Value-guided remapping of sensory cortex by lateral orbitofrontal cortex.

Adaptive behaviour crucially depends on flexible decision-making, which in mammals relies on the frontal cortex, specifically the orbitofrontal cortex (OFC)1-9. How OFC encodes decision variables and instructs sensory areas to guide adaptive behaviour are key open questions. Here we developed a reversal learning task for head-fixed mice, monitored the activity of neurons of the lateral OFC using two-photon calcium imaging and investigated how OFC dynamically interacts with primary somatosensory cortex (S1). Mice learned to discriminate 'go' from 'no-go' tactile stimuli10,11 and adapt their behaviour upon reversal of stimulus-reward contingency ('rule switch'). Imaging individual neurons longitudinally across all behavioural phases revealed a distinct engagement of S1 and lateral OFC, with S1 neural activity reflecting initial task learning, whereas lateral OFC neurons responded saliently and transiently to the rule switch. We identified direct long-range projections from lateral OFC to S1 that can feed this activity back to S1 as value prediction error. This top-down signal updated sensory representations in S1 by functionally remapping responses in a subpopulation of neurons that was sensitive to reward history. Functional remapping crucially depended on top-down feedback as chemogenetic silencing of lateral OFC neurons disrupted reversal learning, as well as plasticity in S1. The dynamic interaction of lateral OFC with sensory cortex thus implements computations critical for value prediction that are history dependent and error based, providing plasticity essential for flexible decision-making.

Context-dependent limb movement encoding in neuronal populations of motor cortex.

Neuronal networks of the mammalian motor cortex (M1) are important for dexterous control of limb joints. Yet it remains unclear how encoding of joint movement in M1 depends on varying environmental contexts. Using calcium imaging we measured neuronal activity in layer 2/3 of the M1 forelimb region while mice grasped regularly or irregularly spaced ladder rungs during locomotion. We found that population coding of forelimb joint movements is sparse and varies according to the flexibility demanded from individual joints in the regular and irregular context, even for equivalent grasping actions across conditions. This context-dependence of M1 encoding emerged during task learning, fostering higher precision of grasping actions, but broke apart upon silencing of projections from secondary motor cortex (M2). These findings suggest that M1 exploits information from M2 to adapt encoding of joint movements to the flexibility demands of distinct familiar contexts, thereby increasing the accuracy of motor output.

The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue.

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).

iDISCO+ for the Study of Neuroimmune Architecture of the Rat Auditory Brainstem.

The lower stations of the auditory system display a complex anatomy. The inner ear labyrinth is composed of several interconnecting membranous structures encased in cavities of the temporal bone, and the cerebellopontine angle contains fragile structures such as meningeal folds, the choroid plexus (CP), and highly variable vascular formations. For this reason, most histological studies of the auditory system have either focused on the inner ear or the CNS by physically detaching the temporal bone from the brainstem. However, several studies of neuroimmune interactions have pinpointed the importance of structures such as meninges and CP; in the auditory system, an immune function has also been suggested for inner ear structures such as the endolymphatic duct (ED) and sac. All these structures are thin, fragile, and have complex 3D shapes. In order to study the immune cell populations located on these structures and their relevance to the inner ear and auditory brainstem in health and disease, we obtained a clarified-decalcified preparation of the rat hindbrain still attached to the intact temporal bone. This preparation may be immunolabeled using a clearing protocol (based on iDISCO+) to show location and functional state of immune cells. The observed macrophage distribution suggests the presence of CP-mediated communication pathways between the inner ear and the cochlear nuclei.

Tissue Clearing and Light Sheet Microscopy: Imaging the Unsectioned Adult Zebra Finch Brain at Cellular Resolution.

The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies.

Ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response.

Brain calcifications are commonly detected in aged individuals and accompany numerous brain diseases, but their functional importance is not understood. In cases of primary familial brain calcification, an autosomally inherited neuropsychiatric disorder, the presence of bilateral brain calcifications in the absence of secondary causes of brain calcification is a diagnostic criterion. To date, mutations in five genes including solute carrier 20 member 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), myogenesis regulating glycosidase (MYORG), platelet-derived growth factor B (PDGFB) and platelet-derived growth factor receptor ß (PDGFRB), are considered causal. Previously, we have reported that mutations in PDGFB in humans are associated with primary familial brain calcification, and mice hypomorphic for PDGFB (Pdgfbret/ret) present with brain vessel calcifications in the deep regions of the brain that increase with age, mimicking the pathology observed in human mutation carriers. In this study, we characterize the cellular environment surrounding calcifications in Pdgfbret/ret animals and show that cells around vessel-associated calcifications express markers for osteoblasts, osteoclasts and osteocytes, and that bone matrix proteins are present in vessel-associated calcifications. Additionally, we also demonstrate the osteogenic environment around brain calcifications in genetically confirmed primary familial brain calcification cases. We show that calcifications cause oxidative stress in astrocytes and evoke expression of neurotoxic astrocyte markers. Similar to previously reported human primary familial brain calcification cases, we describe high interindividual variation in calcification load in Pdgfbret/ret animals, as assessed by ex vivo and in vivo quantification of calcifications. We also report that serum of Pdgfbret/ret animals does not differ in calcification propensity from control animals and that vessel calcification occurs only in the brains of Pdgfbret/ret animals. Notably, ossification of vessels and astrocytic neurotoxic response is associated with specific behavioural and cognitive alterations, some of which are associated with primary familial brain calcification in a subset of patients.

Prion pathogenesis is unaltered in a mouse strain with a permeable blood-brain barrier.

Transmissible spongiform encephalopathies (TSEs) are caused by the prion, which consists essentially of PrPSc, an aggregated, conformationally modified form of the cellular prion protein (PrPC). Although TSEs can be experimentally transmitted by intracerebral inoculation, most instances of infection in the field occur through extracerebral routes. The epidemics of kuru and variant Creutzfeldt-Jakob disease were caused by dietary exposure to prions, and parenteral administration of prion-contaminated hormones has caused hundreds of iatrogenic TSEs. In all these instances, the development of postexposure prophylaxis relies on understanding of how prions propagate from the site of entry to the brain. While much evidence points to lymphoreticular invasion followed by retrograde transfer through peripheral nerves, prions are present in the blood and may conceivably cross the blood-brain barrier directly. Here we have addressed the role of the blood-brain barrier (BBB) in prion disease propagation using Pdgfbret/ret mice which possess a highly permeable BBB. We found that Pdgfbret/ret mice have a similar prion disease incubation time as their littermate controls regardless of the route of prion transmission. These surprising results indicate that BBB permeability is irrelevant to the initiation of prion disease, even when prions are administered parenterally.