Serial two-photon tomography for automated ex vivo mouse brain imaging
TL;DR: An automated method is described that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning, which opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders.
Abstract: Here we describe an automated method, named serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in three dimensions, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders.
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01 Aug 2000
TL;DR: Assessment of medical technology in the context of commercialization with Bioentrepreneur course, which addresses many issues unique to biomedical products.
Abstract: BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.
4,833 citations
TL;DR: A brain-wide, cellular-level, mesoscale connectome for the mouse, using enhanced green fluorescent protein-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain.
Abstract: Comprehensive knowledge of the brain's wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.
2,051 citations
TL;DR: It is shown that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.
Abstract: Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.
1,776 citations
TL;DR: Optogenetic manipulations indicate that the hypothalamus plays an integral role to instantiate emotion states, and is not simply a passive effector of upstream emotion centers.
Abstract: Defensive behaviors reflect underlying emotion states, such as fear. The hypothalamus plays a role in such behaviors, but prevailing textbook views depict it as an effector of upstream emotion centers, such as the amygdala, rather than as an emotion center itself. We used optogenetic manipulations to probe the function of a specific hypothalamic cell type that mediates innate defensive responses. These neurons are sufficient to drive multiple defensive actions, and required for defensive behaviors in diverse contexts. The behavioral consequences of activating these neurons, moreover, exhibit properties characteristic of emotion states in general, including scalability, (negative) valence, generalization and persistence. Importantly, these neurons can also condition learned defensive behavior, further refuting long-standing claims that the hypothalamus is unable to support emotional learning and therefore is not an emotion center. These data indicate that the hypothalamus plays an integral role to instantiate emotion states, and is not simply a passive effector of upstream emotion centers.
921 citations
TL;DR: Protocols spanning multiple dimensions of the CLARITY workflow are described, ranging from simple, reliable and efficient lipid removal without electrophoretic instrumentation to optimized objectives and integration with light-sheet optics (CLARITY-optimized light- sheet microscopy (COLM)) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution.
Abstract: CLARITY enables the chemical transformation of intact biological tissues into a hydrogel–tissue hybrid. The hybrid samples can be interrogated using light and macromolecular labels, whilst retaining fine structure and native biological molecules.
776 citations
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TL;DR: The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation.
Abstract: Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
8,905 citations
TL;DR: An anatomically comprehensive digital atlas containing the expression patterns of ∼20,000 genes in the adult mouse brain is described, providing an open, primary data resource for a wide variety of further studies concerning brain organization and function.
Abstract: Molecular approaches to understanding the functional circuitry of the nervous system promise new insights into the relationship between genes, brain and behaviour. The cellular diversity of the brain necessitates a cellular resolution approach towards understanding the functional genomics of the nervous system. We describe here an anatomically comprehensive digital atlas containing the expression patterns of approximately 20,000 genes in the adult mouse brain. Data were generated using automated high-throughput procedures for in situ hybridization and data acquisition, and are publicly accessible online. Newly developed image-based informatics tools allow global genome-scale structural analysis and cross-correlation, as well as identification of regionally enriched genes. Unbiased fine-resolution analysis has identified highly specific cellular markers as well as extensive evidence of cellular heterogeneity not evident in classical neuroanatomical atlases. This highly standardized atlas provides an open, primary data resource for a wide variety of further studies concerning brain organization and function.
4,944 citations
01 Aug 2000
TL;DR: Assessment of medical technology in the context of commercialization with Bioentrepreneur course, which addresses many issues unique to biomedical products.
Abstract: BIOE 402. Medical Technology Assessment. 2 or 3 hours. Bioentrepreneur course. Assessment of medical technology in the context of commercialization. Objectives, competition, market share, funding, pricing, manufacturing, growth, and intellectual property; many issues unique to biomedical products. Course Information: 2 undergraduate hours. 3 graduate hours. Prerequisite(s): Junior standing or above and consent of the instructor.
4,833 citations
TL;DR: The software consists of a collection of algorithms that are commonly used to solve medical image registration problems, and allows the user to quickly configure, test, and compare different registration methods for a specific application.
Abstract: Medical image registration is an important task in medical image processing. It refers to the process of aligning data sets, possibly from different modalities (e.g., magnetic resonance and computed tomography), different time points (e.g., follow-up scans), and/or different subjects (in case of population studies). A large number of methods for image registration are described in the literature. Unfortunately, there is not one method that works for all applications. We have therefore developed elastix, a publicly available computer program for intensity-based medical image registration. The software consists of a collection of algorithms that are commonly used to solve medical image registration problems. The modular design of elastix allows the user to quickly configure, test, and compare different registration methods for a specific application. The command-line interface enables automated processing of large numbers of data sets, by means of scripting. The usage of elastix for comparing different registration methods is illustrated with three example experiments, in which individual components of the registration method are varied.
3,444 citations
TL;DR: Each of 25 independently generated transgenic lines expressed XFP in a unique pattern, even though all incorporated identical regulatory elements (from the thyl gene), for example, all retinal ganglion cells or many cortical neurons were XFP positive in some lines, whereas only a few ganglions or only layer 5 cortical pyramids were labeled in others.
2,929 citations