Three dimensional reconstruction by electron microscopy in the life sciences: An introduction for cell and tissue biologists

Miranda, Kildare; Girard-Dias, Wendell; Attias, Márcia; Souza, Wanderley de; Ramos, Isabela

doi:10.1002/mrd.22455

Cited by 55 publications

(35 citation statements)

References 94 publications

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“…Within the past 20 years, great progress has been made in EM probe development [26, 27, 35, 38], instrumentation [53, 54, 63], image acquisition [57, 64], image reconstruction [65, 66], and segmentation [67, 68]. In contrast to conventional EM stains in which most objects are not stained specifically, now we have a diverse set of powerful tools to identify a specific protein or structure in EM by localized oxidation of DAB into osmiophilic polymer that selectively contrasts the protein of interest, either through photo-oxidation or enzymatic-oxidation.…”

Section: Discussionmentioning

confidence: 99%

Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy

Deerinck

Bushong

et al. 2015

Methods

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Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host’s cellular environment, their natural in-situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940’s and subsequent application to cells in the 1950’s. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in-situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and preparation of photo-oxidized samples for TEM and serial block-face scanning EM (SBEM) for large-scale volume EM data acquisition are also presented. As an example, we discuss the recent multi-scale analysis of Adenoviral protein E4-ORF3 that reveals a new type of multi-functional polymer that disrupts multiple cellular proteins. This new capability to visualize unambiguously specific viral protein structures at high resolutions in the native cellular environment is revealing new insights into how they usurp host proteins and functions to drive pathological viral replication.

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Section: Discussionmentioning

confidence: 99%

Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy

Deerinck

Bushong

et al. 2015

Methods

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“…While SEM micrographs provide an impressive depth of field, traditionally a given image is limited to a single surface of a tissue section. However, modern SEM approaches can reconstruct a 3‐D volume from a series of 2‐D images made on thin sections of tissue prepared using a microtome or cryostat . One way this process has been streamlined is via serial block‐face scanning electron microscopy (SBFSEM), which combines high‐throughput tissue sectioning and subsequent image acquisition .…”

Section: Imaging Brain Structure At Subcellular Resolution With Scannmentioning

confidence: 99%

“…However, modern SEM approaches can reconstruct a 3-D volume from a series of 2-D images made on thin sections of tissue prepared using a microtome or cryostat. 150,151 One way this process has been streamlined is via serial block-face scanning electron microscopy (SBFSEM), which combines high-throughput tissue sectioning and subsequent image acquisition. 152 In this technique, the surface of a tissue block is imaged with repeated passes of SEM as the top layer of the tissue is shaved away by a microtome to access successively deeper sections.…”

Section: Imaging Brain Structure At Subcellular Resolution With Scannmentioning

confidence: 99%

Mapping causal pathways from genetics to neuropsychiatric disorders using genome‐wide imaging genetics: Current status and future directions

Stein

2019

Psychiatry Clin Neurosci

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Imaging genetics aims to identify genetic variants associated with the structure and function of the human brain. Recently, collaborative consortia have been successful in this goal, identifying and replicating common genetic variants influencing gross human brain structure as measured through magnetic resonance imaging. In this review, we contextualize imaging genetic associations as one important link in understanding the causal chain from genetic variant to increased risk for neuropsychiatric disorders. We provide examples in other fields of how identifying genetic variant associations to disease and multiple phenotypes along the causal chain has revealed a mechanistic understanding of disease risk, with implications for how imaging genetics can be similarly applied. We discuss current findings in the imaging genetics research domain, including that common genetic variants can have a slightly larger effect on brain structure than on risk for disorders like schizophrenia, indicating a somewhat simpler genetic architecture. Also, gross brain structure measurements share a genetic basis with some, but not all, neuropsychiatric disorders, invalidating the previously held belief that they are broad endophenotypes, yet pinpointing brain regions likely involved in the pathology of specific disorders. Finally, we suggest that in order to build a more detailed mechanistic understanding of the effects of genetic variants on the brain, future directions in imaging genetics research will require observations of cellular and synaptic structure in specific brain regions beyond the resolution of magnetic resonance imaging. We expect that integrating genetic associations at biological levels from synapse to sulcus will reveal specific causal pathways impacting risk for neuropsychiatric disorders.

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“…While not commonly used in CLEM studies, it is plausible to envision that some of these additional types of electron microscopy may be beneficial for a particular question. For readers interested in learning more on electron microscopy techniques, we recommend reading one of the many excellent reviews available on this exciting topic (Briggman and Bock, 2012; Knott and Genoud, 2013; Miranda et al, 2015; Perkins et al, 2015). …”

Section: Electron and Light-based Techniques Used For Clemmentioning

confidence: 99%

Correlative Light Electron Microscopy: Connecting Synaptic Structure and Function

Begemann

Galic

2016

Front. Synaptic Neurosci.

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Many core paradigms of contemporary neuroscience are based on information obtained by electron or light microscopy. Intriguingly, these two imaging techniques are often viewed as complementary, yet separate entities. Recent technological advancements in microscopy techniques, labeling tools, and fixation or preparation procedures have fueled the development of a series of hybrid approaches that allow correlating functional fluorescence microscopy data and ultrastructural information from electron micrographs from a singular biological event. As correlative light electron microscopy (CLEM) approaches become increasingly accessible, long-standing neurobiological questions regarding structure-function relation are being revisited. In this review, we will survey what developments in electron and light microscopy have spurred the advent of correlative approaches, highlight the most relevant CLEM techniques that are currently available, and discuss its potential and limitations with respect to neuronal and synapse-specific applications.

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Three dimensional reconstruction by electron microscopy in the life sciences: An introduction for cell and tissue biologists

Cited by 55 publications

References 94 publications

Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy

Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy

Mapping causal pathways from genetics to neuropsychiatric disorders using genome‐wide imaging genetics: Current status and future directions

Correlative Light Electron Microscopy: Connecting Synaptic Structure and Function

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