Serial two-photon tomography for automated ex vivo mouse brain imaging (original) (raw)
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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.
High-Speed and Scalable Whole-Brain Imaging in Rodents and Primates
Neuron
Subcellular resolution imaging of the whole brain and subsequent image analysis are prerequisites for understanding anatomical and functional brain networks. Here, we have developed a very high-speed serial-sectioning imaging system named FAST (block-face serial microscopy tomography), which acquires high-resolution images of a whole mouse brain in a speed range comparable to that of lightsheet fluorescence microscopy. FAST enables complete visualization of the brain at a resolution sufficient to resolve all cells and their subcellular structures. FAST renders unbiased quantitative group comparisons of normal and disease model brain cells for the whole brain at a high spatial resolution. Furthermore, FAST is highly scalable to non-human primate brains and human postmortem brain tissues, and can visualize neuronal projections in a whole adult marmoset brain. Thus, FAST provides new opportunities for global approaches that will allow for a better understanding of brain systems in multiple animal models and in human diseases.
Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high-resolution optical imaging techniques operating in the mm-cm range, ideally suited forex vivo3D whole mouse brain imaging. Although these techniques exhibit high sensitivity and specificity for antibody-labeled targets, the provided anatomical information remains limited. To allow anatomical mapping of fluorescent signal in whole brain, we developed a novel magnetic resonance (MR) – based template with its associated tissue priors and atlas labels, specifically designed for brains subjected to tissue processing protocols required for 3D optical imaging. We investigated the effect of tissue pre-processing and clearing on brain size and morphology and developed optimized templates for BABB/Murrays clear (OCUM) and DBE/iDISCO (iOCUM) cleared brains. By creating optical-(i)OCUM fusion images using our mapping procedure, we localized dopamine transporter and translocator protein expression and tra...
Multi-modal imaging of a single mouse brain over five orders of magnitude of resolution
2020
ABSTRACTMammalian neurons operate at length scales spanning five orders of magnitude; micron-scale-diameter myelinated axons project millimeters across brain regions, ultimately forming nanometer scale synapses on individual post-synaptic neurons. Capturing these anatomical features across that breadth of scale has required imaging samples with multiple independent imaging modalities (e.g. MRI, electron microscopy, etc.). Translating between the different modalities, however, requires imaging the same brain with each. Here, we imaged the same postmortem mouse brain over five orders of spatial resolution using MRI, whole brain micron-scale synchrotron x-ray tomography (μCT), and large volume automated serial electron microscopy. Using this pipeline, we can track individual myelinated axons previously relegated to axon bundles in diffusion tensor MRI or arbitrarily trace neurons and their processes brain-wide and identify individual synapses on them. This pipeline provides both an unp...
Frontiers in Neuroanatomy, 2011
Recent advances in high-throughput technology facilitate massive data collection and sharing, enabling neuroscientists to explore the brain across a large range of spatial scales. One such form of high-throughput data collection is the construction of large-scale mosaic volumes using light microscopy . With this technology, researchers can collect and analyze high-resolution digitized volumes of whole brain sections down to 0.2 μm. However, until recently, scientists lacked the tools to easily handle these large highresolution datasets. Furthermore, artifacts resulting from specimen preparation limited volume reconstruction using this technique to only a single tissue section. In this paper, we carefully describe the steps we used to digitally reconstruct a series of consecutive mouse brain sections labeled with three stains, a stain for blood vessels (DiI), a nuclear stain (TO-PRO-3), and a myelin stain (FluoroMyelin). These stains label important neuroanatomical landmarks that are used for stacking the serial sections during reconstruction. In addition, we show that the use of two software applications, ir-Tweak and Mogrifier, in conjunction with a volume flattening procedure enable scientists to adeptly work with digitized volumes despite tears and thickness variations within tissue sections. These applications make processing large-scale brain mosaics more efficient. When used in combination with new database resources, these brain maps should allow researchers to extend the lifetime of their specimens indefinitely by preserving them in digital form, making them available for future analyses as our knowledge in the field of neuroscience continues to expand.
A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices
Journal of Visualized Experiments, 2011
A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps 1-3 . With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks 4 . Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will 5-6 . Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates 7-8 , alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters 9 . Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
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.
Neuroinformatics
In recent years, the combination of whole-brain immunolabelling, light sheet fluorescence microscopy (LSFM) and subsequent registration of data with a common reference atlas, has enabled 3D visualization and quantification of fluorescent markers or tracers in the adult mouse brain. Today, the common coordinate framework version 3 developed by the Allen’s Institute of Brain Science (AIBS CCFv3), is widely used as the standard brain atlas for registration of LSFM data. However, the AIBS CCFv3 is based on histological processing and imaging modalities different from those used for LSFM imaging and consequently, the data differ in both tissue contrast and morphology. To improve the accuracy and speed by which LSFM-imaged whole-brain data can be registered and quantified, we have created an optimized digital mouse brain atlas based on immunolabelled and solvent-cleared brains. Compared to the AIBS CCFv3 atlas, our atlas resulted in faster and more accurate mapping of neuronal activity as...
Shadow imaging for panoptical visualization of living brain tissue
bioRxiv (Cold Spring Harbor Laboratory), 2022
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, do not have cellular resolution or only give a fragmented view. 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. The human brain is a structural engineering marvel where hundreds of thousands of miles worth of tightly packed axons and dendrites connect billions of neurons into a gigantic electrochemical network that generates memory, thought and action. Its main animal model, the mouse brain, is about three orders of magnitude smaller, but its anatomical structure is similarly dense and complex 1. Fluorescence microscopy is the method of choice for multiscale imaging of living brain tissue with high resolution. However, it typically relies on labeling sparse sets of brain cells, providing a fragmented and partial view of tissue anatomy. Electron microscopy and magnetic resonance imaging are practically label-free and unbiased approaches, yet they either offer high spatial resolution or non-invasive live imaging, but not both together. Moreover, they cannot be combined in situ with other powerful neuro-technologies, such as Ca 2+ imaging, electrophysiology and optogenetics. Getting a more detailed and broader view of brain tissue is not only important for mapping the functional connectivity of neuronal circuits 2 , it can also unearth useful information on anatomical context and tissue viability to assist in experiments and their interpretation.