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1.
IEEE Trans Med Imaging ; PP2022 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-35377840

RESUMO

As connectomic datasets exceed hundreds of terabytes in size, accurate and efficient skeleton generation of the label volumes has evolved into a critical component of the computation pipeline used for analysis, evaluation, visualization, and error correction. We propose a novel topological thinning strategy that uses biological-constraints to produce accurate centerlines from segmented neuronal volumes while still maintaining biologically relevant properties. Current methods are either agnostic to the underlying biology, have non-linear running times as a function of the number of input voxels, or both. First, we eliminate from the input segmentation biologically-infeasible bubbles, pockets of voxels incorrectly labeled within a neuron, to improve segmentation accuracy, allow for more accurate centerlines, and increase processing speed. Next, a Convolutional Neural Network (CNN) detects cell bodies from the input segmentation, allowing us to anchor our skeletons to the somata. Lastly, a synapse-aware topological thinning approach produces expressive skeletons for each neuron with a nearly one-to-one correspondence between endpoints and synapses. We simultaneously estimate geometric properties of neurite width and geodesic distance between synapse and cell body, improving accuracy by 47.5% and 62.8% over baseline methods. We separate the skeletonization process into a series of computation steps, leveraging data-parallel strategies to increase throughput significantly. We demonstrate our results on over 1250 neurons and neuron fragments from three different species, processing over one million voxels per second per CPU with linear scalability.

2.
Curr Biol ; 32(1): 176-189.e5, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-34822765

RESUMO

All animals need to differentiate between exafferent stimuli, which are caused by the environment, and reafferent stimuli, which are caused by their own movement. In the case of mechanosensation in aquatic animals, the exafferent inputs are water vibrations in the animal's proximity, which need to be distinguishable from the reafferent inputs arising from fluid drag due to locomotion. Both of these inputs are detected by the lateral line, a collection of mechanosensory organs distributed along the surface of the body. In this study, we characterize in detail how hair cells-the receptor cells of the lateral line-in zebrafish larvae discriminate between such reafferent and exafferent signals. Using dye labeling of the lateral line nerve, we visualize two parallel descending inputs that can influence lateral line sensitivity. We combine functional imaging with ultra-structural EM circuit reconstruction to show that cholinergic signals originating from the hindbrain transmit efference copies (copies of the motor command that cancel out self-generated reafferent stimulation during locomotion) and that dopaminergic signals from the hypothalamus may have a role in threshold modulation, both in response to locomotion and salient stimuli. We further gain direct mechanistic insight into the core components of this circuit by loss-of-function perturbations using targeted ablations and gene knockouts. We propose that this simple circuit is the core implementation of mechanosensory reafferent suppression in these young animals and that it might form the first instantiation of state-dependent modulation found at later stages in development.


Assuntos
Sistema da Linha Lateral , Peixe-Zebra , Animais , Larva , Sistema da Linha Lateral/fisiologia , Locomoção/fisiologia , Rombencéfalo , Peixe-Zebra/fisiologia
3.
Nature ; 596(7871): 257-261, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349261

RESUMO

An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.


Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Caenorhabditis elegans/citologia , Conectoma , Modelos Neurológicos , Vias Neurais , Sinapses/fisiologia , Envelhecimento/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/ultraestrutura , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/ultraestrutura , Individualidade , Interneurônios/citologia , Microscopia Eletrônica , Neurônios/citologia , Comportamento Estereotipado
4.
Neuroimage ; 243: 118530, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34464739

RESUMO

The first phase of the Human Connectome Project pioneered advances in MRI technology for mapping the macroscopic structural connections of the living human brain through the engineering of a whole-body human MRI scanner equipped with maximum gradient strength of 300 mT/m, the highest ever achieved for human imaging. While this instrument has made important contributions to the understanding of macroscale connectional topology, it has also demonstrated the potential of dedicated high-gradient performance scanners to provide unparalleled in vivo assessment of neural tissue microstructure. Building on the initial groundwork laid by the original Connectome scanner, we have now embarked on an international, multi-site effort to build the next-generation human 3T Connectome scanner (Connectome 2.0) optimized for the study of neural tissue microstructure and connectional anatomy across multiple length scales. In order to maximize the resolution of this in vivo microscope for studies of the living human brain, we will push the diffusion resolution limit to unprecedented levels by (1) nearly doubling the current maximum gradient strength from 300 mT/m to 500 mT/m and tripling the maximum slew rate from 200 T/m/s to 600 T/m/s through the design of a one-of-a-kind head gradient coil optimized to minimize peripheral nerve stimulation; (2) developing high-sensitivity multi-channel radiofrequency receive coils for in vivo and ex vivo human brain imaging; (3) incorporating dynamic field monitoring to minimize image distortions and artifacts; (4) developing new pulse sequences to integrate the strongest diffusion encoding and highest spatial resolution ever achieved in the living human brain; and (5) calibrating the measurements obtained from this next-generation instrument through systematic validation of diffusion microstructural metrics in high-fidelity phantoms and ex vivo brain tissue at progressively finer scales with accompanying diffusion simulations in histology-based micro-geometries. We envision creating the ultimate diffusion MRI instrument capable of capturing the complex multi-scale organization of the living human brain - from the microscopic scale needed to probe cellular geometry, heterogeneity and plasticity, to the mesoscopic scale for quantifying the distinctions in cortical structure and connectivity that define cyto- and myeloarchitectonic boundaries, to improvements in estimates of macroscopic connectivity.


Assuntos
Conectoma/métodos , Imagem de Difusão por Ressonância Magnética/métodos , Encéfalo/diagnóstico por imagem , Feminino , Humanos , Masculino , Neuroimagem/métodos , Imagens de Fantasmas
5.
Dev Neurobiol ; 81(5): 746-757, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33977655

RESUMO

Dendritic spines are membranous protrusions that receive essentially all excitatory inputs in most mammalian neurons. Spines, with a bulbous head connected to the dendrite by a thin neck, have a variety of morphologies that likely impact their functional properties. Nevertheless, the question of whether spines belong to distinct morphological subtypes is still open. Addressing this quantitatively requires clear identification and measurements of spine necks. Recent advances in electron microscopy enable large-scale systematic reconstructions of spines with nanometer precision in 3D. Analyzing ultrastructural reconstructions from mouse neocortical neurons with computer vision algorithms, we demonstrate that the vast majority of spine structures can be rigorously separated into heads and necks, enabling morphological measurements of spine necks. We then used a database of spine morphological parameters to explore the potential existence of different spine classes. Without exception, our analysis revealed unimodal distributions of individual morphological parameters of spine heads and necks, without evidence for subtypes of spines. The postsynaptic density size was strongly correlated with the spine head volume. The spine neck diameter, but not the neck length, was also correlated with the head volume. Spines with larger head volumes often had a spine apparatus and pairs of spines in a post-synaptic cell contacted by the same axon had similar head volumes. Our data reveal a lack of morphological subtypes of spines and indicate that the spine neck length and head volume must be independently regulated. These results have repercussions for our understanding of the function of dendritic spines in neuronal circuits.


Assuntos
Espinhas Dendríticas , Neurônios , Animais , Axônios/ultraestrutura , Dendritos/fisiologia , Espinhas Dendríticas/fisiologia , Mamíferos , Camundongos , Microscopia Eletrônica , Neurônios/fisiologia , Sinapses
7.
Artigo em Inglês | MEDLINE | ID: mdl-35128463

RESUMO

Tissue clearing of gross anatomical samples was first described over a century ago and has only recently found widespread use in the field of microscopy. This renaissance has been driven by the application of modern knowledge of optical physics and chemical engineering to the development of robust and reproducible clearing techniques, the arrival of new microscopes that can image large samples at cellular resolution and computing infrastructure able to store and analyze large data volumes. Many biological relationships between structure and function require investigation in three dimensions and tissue clearing therefore has the potential to enable broad discoveries in the biological sciences. Unfortunately, the current literature is complex and could confuse researchers looking to begin a clearing project. The goal of this Primer is to outline a modular approach to tissue clearing that allows a novice researcher to develop a customized clearing pipeline tailored to their tissue of interest. Further, the Primer outlines the required imaging and computational infrastructure needed to perform tissue clearing at scale, gives an overview of current applications, discusses limitations and provides an outlook on future advances in the field.

8.
Med Image Comput Comput Assist Interv ; 12265: 66-76, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33283212

RESUMO

Electron microscopy (EM) allows the identification of intracellular organelles such as mitochondria, providing insights for clinical and scientific studies. However, public mitochondria segmentation datasets only contain hundreds of instances with simple shapes. It is unclear if existing methods achieving human-level accuracy on these small datasets are robust in practice. To this end, we introduce the MitoEM dataset, a 3D mitochondria instance segmentation dataset with two (30µm)3 volumes from human and rat cortices respectively, 3, 600× larger than previous benchmarks. With around 40K instances, we find a great diversity of mitochondria in terms of shape and density. For evaluation, we tailor the implementation of the average precision (AP) metric for 3D data with a 45× speedup. On MitoEM, we find existing instance segmentation methods often fail to correctly segment mitochondria with complex shapes or close contacts with other instances. Thus, our MitoEM dataset poses new challenges to the field. We release our code and data: https://donglaiw.github.io/page/mitoEM/index.html.

9.
Comput Vis ECCV ; 12363: 103-120, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33345257

RESUMO

For large-scale vision tasks in biomedical images, the labeled data is often limited to train effective deep models. Active learning is a common solution, where a query suggestion method selects representative unlabeled samples for annotation, and the new labels are used to improve the base model. However, most query suggestion models optimize their learnable parameters only on the limited labeled data and consequently become less effective for the more challenging unlabeled data. To tackle this, we propose a two-stream active query suggestion approach. In addition to the supervised feature extractor, we introduce an unsupervised one optimized on all raw images to capture diverse image features, which can later be improved by fine-tuning on new labels. As a use case, we build an end-to-end active learning framework with our query suggestion method for 3D synapse detection and mitochondria segmentation in connectomics. With the framework, we curate, to our best knowledge, the largest connectomics dataset with dense synapses and mitochondria annotation. On this new dataset, our method outperforms previous state-of-the-art methods by 3.1% for synapse and 3.8% for mitochondria in terms of region-of-interest proposal accuracy. We also apply our method to image classification, where it outperforms previous approaches on CIFAR-10 under the same limited annotation budget. The project page is https://zudi-lin.github.io/projects/#two_stream_active.

10.
Cell ; 182(6): 1372-1376, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32946777

RESUMO

Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.


Assuntos
Encéfalo/fisiologia , Conectoma/métodos , Rede Nervosa/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Camundongos
11.
Nat Protoc ; 15(9): 2773-2784, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32737465

RESUMO

Spherical aberration (SA) occurs when light rays entering at different points of a spherical lens are not focused to the same point of the optical axis. SA that occurs inside the lens elements of a fluorescence microscope is well understood and corrected for. However, SA is also induced when light passes through an interface of refractive index (RI)-mismatched substances (i.e., a discrepancy between the RI of the immersion medium and the RI of the sample). SA due to RI mismatches has many deleterious effects on imaging. Perhaps most important for 3D imaging is that the distance the image plane moves in a sample is not equivalent to the distance traveled by an objective (or stage) during z-stack acquisition. This non-uniform translation along the z axis gives rise to artifactually elongated images (if the objective is immersed in a medium with a higher RI than that of the sample) or compressed images (if the objective is immersed in a medium with a lower RI than that of the sample) and alters the optimal axial sampling rate. In this tutorial, we describe why this distortion occurs, how it impacts quantitative measurements and axial resolution, and what can be done to avoid SA and thereby prevent distorted images. In addition, this tutorial aims to better inform researchers of how to correct RI mismatch-induced axial distortions and provides a practical ImageJ/Fiji-based tool to reduce the prevalence of volumetric measurement errors and lost axial resolution.


Assuntos
Artefatos , Imageamento Tridimensional , Microscopia de Fluorescência , Espalhamento de Radiação , Corantes Fluorescentes/química , Microesferas
12.
Proc Natl Acad Sci U S A ; 117(31): 18780-18787, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32699144

RESUMO

Macular telangiectasia type 2 (MacTel), a late-onset macular degeneration, has been linked to a loss in the retina of Müller glial cells and the amino acid serine, synthesized by the Müller cells. The disease is confined mainly to a central retinal region called the MacTel zone. We have used electron microscopic connectomics techniques, optimized for disease analysis, to study the retina from a 48-y-old woman suffering from MacTel. The major observations made were specific changes in mitochondrial structure within and outside the MacTel zone that were present in all retinal cell types. We also identified an abrupt boundary of the MacTel zone that coincides with the loss of Müller cells and macular pigment. Since Müller cells synthesize retinal serine, we propose that a deficiency of serine, required for mitochondrial maintenance, causes mitochondrial changes that underlie MacTel development.


Assuntos
Conectoma/métodos , Retina , Doenças Retinianas , Feminino , Humanos , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/patologia , Microscopia Eletrônica , Pessoa de Meia-Idade , Retina/citologia , Retina/diagnóstico por imagem , Retina/patologia , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/patologia
13.
Dev Cell ; 53(5): 577-588.e7, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32516597

RESUMO

Axons connect neurons together, establishing the wiring architecture of neuronal networks. Axonal connectivity is largely built during embryonic development through highly constrained processes of axon guidance, which have been extensively studied. However, the inability to control axon guidance, and thus neuronal network architecture, has limited investigation of how axonal connections influence subsequent development and function of neuronal networks. Here, we use zebrafish motor neurons expressing a photoactivatable Rac1 to co-opt endogenous growth cone guidance machinery to precisely and non-invasively direct axon growth using light. Axons can be guided over large distances, within complex environments of living organisms, overriding competing endogenous signals and redirecting axons across potent repulsive barriers to construct novel circuitry. Notably, genetic axon guidance defects can be rescued, restoring functional connectivity. These data demonstrate that intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity, allowing investigation of neural network dynamics in intact living organisms.


Assuntos
Orientação de Axônios , Neurônios Motores/citologia , Optogenética/métodos , Proteínas de Peixe-Zebra/genética , Proteínas rac1 de Ligação ao GTP/genética , Animais , Células Cultivadas , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Sinapses/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
14.
Neuron ; 106(3): 468-481.e2, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32142646

RESUMO

One way to assess a neuron's function is to describe all its inputs and outputs. With this goal in mind, we used serial section electron microscopy to map 899 synaptic inputs and 623 outputs in one inhibitory interneuron in a large volume of the mouse visual thalamus. This neuron innervated 256 thalamocortical cells spread across functionally distinct subregions of the visual thalamus. All but one of its neurites were bifunctional, innervating thalamocortical and local interneurons while also receiving synapses from the retina. We observed a wide variety of local synaptic motifs. While this neuron innervated many cells weakly, with single en passant synapses, it also deployed specialized branches that climbed along other dendrites to form strong multi-synaptic connections with a subset of partners. This neuron's diverse range of synaptic relationships allows it to participate in a mix of global and local processing but defies assigning it a single circuit function.


Assuntos
Interneurônios/fisiologia , Inibição Neural , Sinapses/fisiologia , Tálamo/citologia , Córtex Visual/citologia , Animais , Interneurônios/citologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Técnicas de Rastreamento Neuroanatômico , Tálamo/fisiologia , Córtex Visual/fisiologia
15.
Cell Rep ; 29(9): 2849-2861.e6, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31775050

RESUMO

During postnatal development, cerebellar climbing fibers alter their innervation strengths onto supernumerary Purkinje cell targets, generating a one-to-few connectivity pattern in adulthood. To get insight about the processes responsible for this remapping, we reconstructed serial electron microscopy datasets from mice during the first postnatal week. Between days 3 and 7, individual climbing fibers selectively add many synapses onto a subset of Purkinje targets in a positive-feedback manner, without pruning synapses from other targets. Active zone sizes of synapses associated with powerful versus weak inputs are indistinguishable. Changes in synapse number are thus the predominant form of early developmental plasticity. Finally, the numbers of climbing fibers and Purkinje cells in a local region nearly match. Initial over-innervation of Purkinje cells by climbing fibers is therefore economical: the number of axons entering a region is enough to assure that each ultimately retains a postsynaptic target and that none branched there in vain.


Assuntos
Cerebelo/fisiopatologia , Fibras Nervosas/metabolismo , Sinapses/metabolismo , Animais , Humanos , Camundongos
16.
J Neurosci ; 39(42): 8275-8284, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619497

RESUMO

The overarching goal of the NIH BRAIN (Brain Research through Advancing Innovative Neurotechnologies) Initiative is to advance the understanding of healthy and diseased brain circuit function through technological innovation. Core principles for this goal include the validation and dissemination of the myriad innovative technologies, tools, methods, and resources emerging from BRAIN-funded research. Innovators, BRAIN funding agencies, and non-Federal partners are working together to develop strategies for making these products usable, available, and accessible to the scientific community. Here, we describe several early strategies for supporting the dissemination of BRAIN technologies. We aim to invigorate a dialogue with the neuroscience research and funding community, interdisciplinary collaborators, and trainees about the existing and future opportunities for cultivating groundbreaking research products into mature, integrated, and adaptable research systems. Along with the accompanying Society for Neuroscience 2019 Mini-Symposium, "BRAIN Initiative: Cutting-Edge Tools and Resources for the Community," we spotlight the work of several BRAIN investigator teams who are making progress toward providing tools, technologies, and services for the neuroscience community. These tools access neural circuits at multiple levels of analysis, from subcellular composition to brain-wide network connectivity, including the following: integrated systems for EM- and florescence-based connectomics, advances in immunolabeling capabilities, and resources for recording and analyzing functional connectivity. Investigators describe how the resources they provide to the community will contribute to achieving the goals of the NIH BRAIN Initiative. Finally, in addition to celebrating the contributions of these BRAIN-funded investigators, the Mini-Symposium will illustrate the broader diversity of BRAIN Initiative investments in cutting-edge technologies and resources.


Assuntos
Neurociências/métodos , Pesquisa , Tecnologia , Humanos
17.
Methods Cell Biol ; 152: 41-67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31326026

RESUMO

The Automated Tape-Collecting Ultramicrotome (ATUM) is a tape-reeling device that is placed in a water-filled diamond knife boat to collect serial sections as they are cut by a conventional ultramicrotome. The ATUM can collect thousands of sections of many different shapes and sizes, which are subsequently imaged by a scanning electron microscope. This method has been used for large-scale connectomics projects of mouse brain, and is well suited for other smaller-scale studies of tissues, cells, and organisms. Here, we describe basic procedures for preparing a block for ATUM sectioning, handling of the ATUM, tape preparation, post-treatment of sections, and considerations for mapping, imaging, and aligning the serial sections.


Assuntos
Microscopia Eletrônica de Varredura/métodos , Microtomia/métodos , Animais , Encéfalo/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Camundongos
18.
Cell Rep ; 27(10): 2799-2808.e3, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167127

RESUMO

Reciprocal communication between neurons and oligodendrocytes is essential for the generation and localization of myelin, a critical feature of the CNS. In the neocortex, individual oligodendrocytes can myelinate multiple axons; however, the neuronal origin of the myelinated axons has remained undefined and, while largely assumed to be from excitatory pyramidal neurons, it also includes inhibitory interneurons. This raises the question of whether individual oligodendrocytes display bias for the class of neurons that they myelinate. Here, we find that different classes of cortical interneurons show distinct patterns of myelin distribution starting from the onset of myelination, suggesting that oligodendrocytes can recognize the class identity of individual types of interneurons that they target. Notably, we show that some oligodendrocytes disproportionately myelinate the axons of inhibitory interneurons, whereas others primarily target excitatory axons or show no bias. These results point toward very specific interactions between oligodendrocytes and neurons and raise the interesting question of why myelination is differentially directed toward different neuron types.


Assuntos
Axônios/metabolismo , Bainha de Mielina/fisiologia , Neocórtex/fisiologia , Oligodendroglia/metabolismo , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Feminino , Interneurônios/citologia , Interneurônios/metabolismo , Interneurônios/fisiologia , Interneurônios/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Bainha de Mielina/metabolismo , Neocórtex/metabolismo , Neocórtex/ultraestrutura , Inibição Neural , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Oligodendroglia/ultraestrutura , Células Piramidais/metabolismo , Software
19.
J Surg Res ; 242: 207-213, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31085369

RESUMO

BACKGROUND: Peripheral nerve assessment has traditionally been studied through histological and immunological staining techniques in a limited cross-sectional modality, making detailed analysis difficult. A new application of serial section electron microscopy is presented to overcome these limitations. METHODS: Direct nerve repairs were performed on the posterior auricular nerve of transgenic YFP-H mice. Six weeks postoperatively the nerves were imaged using confocal fluorescent microscopy then excised and embedded in resin. Resin blocks were sequentially sectioned at 100 nm, and sections were serially imaged with an electron microscope. Images were aligned and autosegmented to allow for 3D reconstruction. RESULTS: Basic morphometry and axonal counts were fully automated. Using full 3D reconstructions, the relationships between the axons, the Nodes of Ranvier, and Schwann cells could be fully appreciated. Interactions of individual axons with their surrounding environment could be visualized and explored in a virtual three-dimensional space. CONCLUSIONS: Serial section electron microscopy allows the detailed pathway of the regenerating axon to be visualized in a 3D virtual space in comparison to isolated individual traditional histological techniques. Fully automated histo-morphometry can now give accurate axonal counts, provide information regarding the quality of nerve regeneration, and reveal the cell-to-cell interaction at a super-resolution scale. It is possible to fully visualize and "fly-through" the nerve to help understand the behavior of a regenerating axon within its environment. This technique provides future opportunities to evaluate the effect different treatment modalities have on the neuroregenerative potential and help us understand the impact different surgical techniques have when treating nerve injuries.


Assuntos
Axônios/fisiologia , Imageamento Tridimensional/métodos , Microscopia Intravital/métodos , Regeneração Nervosa , Nervos Periféricos/diagnóstico por imagem , Animais , Axônios/ultraestrutura , Proteínas de Bactérias/genética , Comunicação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Corantes Fluorescentes , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Procedimentos Neurocirúrgicos/métodos , Traumatismos dos Nervos Periféricos/diagnóstico por imagem , Traumatismos dos Nervos Periféricos/cirurgia , Nervos Periféricos/fisiologia , Nervos Periféricos/cirurgia , Nós Neurofibrosos/fisiologia , Nós Neurofibrosos/ultraestrutura
20.
Front Neural Circuits ; 13: 29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31133819

RESUMO

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.


Assuntos
Encéfalo/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Neuroimagem/métodos , Animais , Callithrix , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos
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