Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution (original) (raw)
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Fast wide-volume functional imaging of engineered in vitro brain tissues
Scientific Reports
The need for in vitro models that mimic the human brain to replace animal testing and allow highthroughput screening has driven scientists to develop new tools that reproduce tissue-like features on a chip. Three-dimensional (3D) in vitro cultures are emerging as an unmatched platform that preserves the complexity of cell-to-cell connections within a tissue, improves cell survival, and boosts neuronal differentiation. In this context, new and flexible imaging approaches are required to monitor the functional states of 3D networks. Herein, we propose an experimental model based on 3D neuronal networks in an alginate hydrogel, a tunable wide-volume imaging approach, and an efficient denoising algorithm to resolve, down to single cell resolution, the 3D activity of hundreds of neurons expressing the calcium sensor GCaMP6s. Furthermore, we implemented a 3D co-culture system mimicking the contiguous interfaces of distinct brain tissues such as the cortical-hippocampal interface. The analysis of the network activity of single and layered neuronal co-cultures revealed cell-type-specific activities and an organization of neuronal subpopulations that changed in the two culture configurations. Overall, our experimental platform represents a simple, powerful and cost-effective platform for developing and monitoring living 3D layered brain tissue on chip structures with high resolution and high throughput. The design and development of realistic experimental models of the human brain remain major constraints in studying nervous system functions in health and disease, developing new treatment strategies, and testing prosthetic devices 1. For these reasons, in vitro models represent an attractive and ethical tool since they allow high-throughput cost-accessible investigation 2. Moreover, the development of cell reprogramming and differentiation protocols that allow investigators to recapitulate human diseases in a dish further discourages the use of animal models. The most common approach to building a brain on a chip is based on 2D cultures of defined neural cell types with a controlled network topology 3. These models are highly reproducible and are compatible with standard monitoring systems 4. However, growth of cells on a 2D support alters cellular physiology 5 and fails to reproduce the real properties of a tissue. Growth on stiff Petri dish surfaces induces aberrant branching of neuronal processes 6 , which, together with the lower degree of freedom of neurite navigation in a 2D setting, typically promotes overloading of interneuronal connections. This, in turn, may cause the observed amplification of the activity of 2D neuronal networks on stiff substrates 7 , which may lead to non-physiological states 8. In this regard, it is widely recognized that mechanical and topographical cues influence cell survival, proliferation and differentiation and finally the development of functional neural networks 9. Indeed, the architecture of a 3D scaffold allows isotropic distribution of adhesion contacts, thereby modifying the intracellular distribution of mechanical tension and consequently nuclear shape, chromatin arrangement and gene expression in cells 10. Moreover, mechanotaxis influences the motility of neuronal processes 6 , and it has a critical role in axonal pathfinding in vivo 11. For all of these reasons, there has been a growing demand for new technologies and materials (either natural or synthetic)
Multi-Scale Light-Sheet Fluorescence Microscopy for Fast Whole Brain Imaging
Frontiers in Neuroanatomy, 2021
Whole-brain imaging has become an increasingly important approach to investigate neural structures, such as somata distribution, dendritic morphology, and axonal projection patterns. Different structures require whole-brain imaging at different resolutions. Thus, it is highly desirable to perform whole-brain imaging at multiple scales. Imaging a complete mammalian brain at synaptic resolution is especially challenging, as it requires continuous imaging from days to weeks because of the large number of voxels to sample, and it is difficult to acquire a constant quality of imaging because of light scattering during in toto imaging. Here, we reveal that light-sheet microscopy has a unique advantage over wide-field microscopy in multi-scale imaging because of its decoupling of illumination and detection. Based on this observation, we have developed a multi-scale light-sheet microscope that combines tiling of light-sheet, automatic zooming, periodic sectioning, and tissue expansion to ac...
Journal of Visualized Experiments, 2013
Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.
Mesoscopic Imaging: Shining a Wide Light on Large-Scale Neural Dynamics
Neuron, 2020
Optical imaging has revolutionized our ability to monitor brain activity, spanning spatial scales from synapses to cells to circuits. Here, we summarize the rapid development and application of mesoscopic imaging, a widefield fluorescence-based approach that balances high spatiotemporal resolution with extraordinarily large fields of view. By leveraging the continued expansion of fluorescent reporters for neuronal activity and novel strategies for indicator expression, mesoscopic analysis enables measurement and correlation of network dynamics with behavioral state and task performance. Moreover, the combination of widefield imaging with cellular resolution methods such as two-photon microscopy and electrophysiology is bridging boundaries between cellular and network analyses. Overall, mesoscopic imaging provides a powerful option in the optical toolbox for investigation of brain function.
Large-scale cellular-resolution imaging of neural activity in freely behaving mice
2021
Miniaturized microscopes for head-mounted fluorescence imaging are powerful tools for visualizing neural activity during naturalistic behaviors, but the restricted field of view of first-generation ‘miniscopes’ limits the size of neural populations accessible for imaging. Here we describe a novel miniaturized mesoscope offering cellular-resolution imaging over areas spanning several millimeters in freely moving mice. This system enables comprehensive visualization of activity across entire brain regions or interactions across areas.
2021
Cells are not uniformly distributed in the human cerebral cortex. Rather, they are arranged in a regional and laminar fashion that span a range of scales. Here we demonstrate an innovative imaging and analysis pipeline to construct a reliable cell census across the human cerebral cortex. Magnetic resonance imaging (MRI) is used to establish a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with both traditional immunohistochemistry, to provide a stereological gold-standard, and with a custom-made inverted confocal light-sheet fluorescence microscope (LSFM) for 3D imaging at cellular resolution. Finally, mesoscale optical coherence tomography (OCT) enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system.
Biomedical Optics Express, 2021
Although neuronal density analysis on human brain slices is available from stereological studies, data on the spatial distribution of neurons in 3D are still missing. Since the neuronal organization is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than relying on sparse sampling methods. To achieve this goal, we implement a new tissue transformation protocol to clear and label human brain tissues and we exploit the high-resolution optical sectioning of two-photon fluorescence microscopy to perform 3D mesoscopic reconstruction. We perform neuronal mapping of 100mm3 human brain samples and evaluate the volume and density distribution of neurons from various areas of the cortex originating from different subjects (young, adult, and elderly, both healthy and pathological). The quantitative evaluation of the density in combination with the mean volume of the thousands of neurons identified within the specimens, allow us to determine...
Miniaturized device for whole cortex mesoscale imaging in freely behaving mice
2020
The advent of genetically-encoded calcium indicators, along with surgical preparations such as thinned skulls or refractive index matched skulls, have enabled mesoscale cortical activity imaging in headfixed mice. Such imaging studies have revealed complex patterns of coordinated activity across the cortical surface during a variety of behaviors. However, neural activity during free, unrestrained behavior significantly differs from that observed in headfixed animals. The ability to perform mesoscale imaging of the cortex in freely behaving mice may launch new avenues of scientific enquiry. Here we present the ‘Mesoscope’, a miniature, head-mountable imaging device compatible with transparent polymer skulls recently developed by our group. With an 8x10 mm field of view, the Mesoscope can image most of the mouse dorsal cortex and weighs only 3.7 grams. Here, we show preliminary behavioral experiments and mesoscale imaging in anesthetized mice.
Shadow imaging for panoptical visualization of brain tissue in vivo
Nature Communications
Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward ‘shadow imaging’ approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. ...