X-Ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-Face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

Bushong, Eric A.; Johnson, Donald D.; Kim, Keun‐Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T.; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H.

doi:10.1017/s1431927614013579

Cited by 70 publications

(75 citation statements)

References 23 publications

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“…in electron microscopy have greatly facilitated this task (33)(34)(35)(36)(37) and these advances are being extensively exploited for "connectomics" studies (https://www.neuroscienceblueprint.nih.gov/ connectome/) aimed at mapping neuronal wiring in the brain. Here, we have used one such method, focused ion beam-scanning electron microscopy (FIB-SEM), to map the ER and its connections to other membranes within neurons.…”

Section: Significancementioning

confidence: 99%

Contacts between the endoplasmic reticulum and other membranes in neurons

Whiteus

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

428

501

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Close appositions between the membrane of the endoplasmic reticulum (ER) and other intracellular membranes have important functions in cell physiology. These include lipid homeostasis, regulation of Ca 2+ dynamics, and control of organelle biogenesis and dynamics. Although these membrane contacts have previously been observed in neurons, their distribution and abundance have not been systematically analyzed. Here, we have used focused ion beam-scanning electron microscopy to generate 3D reconstructions of intracellular organelles and their membrane appositions involving the ER (distance ≤30 nm) in different neuronal compartments. ER-plasma membrane (PM) contacts were particularly abundant in cell bodies, with large, flat ER cisternae apposed to the PM, sometimes with a notably narrow lumen (thin ER). Smaller ER-PM contacts occurred throughout dendrites, axons, and in axon terminals. ER contacts with mitochondria were abundant in all compartments, with the ER often forming a network that embraced mitochondria. Small focal contacts were also observed with tubulovesicular structures, likely to be endosomes, and with sparse multivesicular bodies and lysosomes found in our reconstructions. Our study provides an anatomical reference for interpreting information about interorganelle communication in neurons emerging from functional and biochemical studies.FIB-SEM | thin ER | Stim1 | spine apparatus | subsurface cisternae

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Section: Significancementioning

confidence: 99%

Contacts between the endoplasmic reticulum and other membranes in neurons

Whiteus

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

428

501

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“…The use of micro-CT microscopy proved a powerful tool for the accurate relocation of the ROI -a central vein that acted as a morphological landmark -as a consequence of the sample being rendered opaque to light, proceeding sample preparation for EM. This practice further ensured that a greater volume of the sample could be collected in the SBF-SEM, due to difficulty in deciding where to capture data from the sample block face{Karreman et al 49 #69; Handschuh et al 50 #67; Bushong et al 51 #66}. Whilst micro-CT was simply used as a relocation tool, future studies could plausibly exploit the technique to further extract relevant structural information.…”

Section: Discussionmentioning

confidence: 99%

3‐D EM exploration of the hepatic microarchitecture ‐ lessons learned for large‐volume in situ serial sectioning

Shami

Cheng

Huynh

et al. 2016

European Microscopy Congress 2016: Proceedings

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To-date serial block-face scanning electron microscopy (SBF-SEM) dominates as the premier technique for generating three-dimensional (3-D) data of resin-embedded biological samples at an unprecedented depth volume. Given the infancy of the technique, limited literature is currently available regarding the applicability of SBF-SEM for the ultrastructural investigation of tissues. Herein, we provide a comprehensive and rigorous appraisal of five different SBF-SEM sample preparation protocols for the large-volume exploration of the hepatic microarchitecture at an unparalleled X, Y and Z resolution. In so doing, we qualitatively and quantitatively validate the use of a comprehensive SBF-SEM sample preparation protocol, based on the application of heavy metal fixatives, stains and mordanting agents. Employing the best-tested SBF-SEM approach, enabled us to assess large-volume morphometric data on murine parenchymal cells, sinusoids and bile canaliculi. Finally, we integrated the validated SBF-SEM protocol with a correlative light and electron microscopy (CLEM) approach. The combination of confocal scanning laser microscopy and SBF-SEM provided a novel way to picture subcellular detail. We appreciate that this multidimensional approach will aid the subsequent research of liver tissue under relevant experimental and disease conditions. 4 #19}, heralded a new era in analysing the complex geometric arrangement of biological cells and tissues at the nanometre scale. Throughout the infancy of EM techniques, including both transmission (TEM) and scanning electron microscopy (SEM), the structural information gained was limited to two-dimensional (2-D) space, providing a mere snapshot of innately three-dimensional (3-D) structures.Throughout the mid-1950s, advances in ultramicrotome design and specimen preparation procedures{Porter and Blum 5 #25} facilitated the generation of 3-D ultrastructural information via the superimposition of consecutive 2-D TEM micrographs that were acquired from a serially sectioned specimen -a technique dubbed serial-section TEM (ssTEM){#387 }. Currently, ssTEM is regarded as a partial effort in the generation of 3-D information, primarily due to the fact that it is a highly arduous and labour-intensive technique that is riddled with several sources of error, such as section compression and the loss of serial sections, which ultimately results in an incomplete data set. The tediousness of the technique is further compounded by the necessity to align image stacks in order to correct for misalignments that occur during the manual imaging of successive sections{Williams and Kallman 12 #15}. Moreover, the generation of high-resolution axial resolution is

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“…This opportunity is provided by X-ray micro-computed tomography (microCT), which uses the presence of heavy metals, such as osmium -and which are required for EM -to resolve a number of anatomical structures, such as the vasculature, hair follicles and collagen fibers (Karreman et al, 2016). This approach has been used on brain vibratome sections and allowed to locate photo-oxidized neuronal structures (Bushong et al, 2015). Moreover, microCT can be used on voluminous samples, such as brain biopsies, and allows to accurately resolve the brain capillary architecture.…”

Section: Ground-breaking In Vitro Clemmentioning

confidence: 99%

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

Follain

Mercier

Osmani

et al. 2016

Journal of Cell Science

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Life is driven by a set of biological events that are naturally dynamic and tightly orchestrated from the single molecule to entire organisms. Although biochemistry and molecular biology have been essential in deciphering signaling at a cellular and organismal level, biological imaging has been instrumental for unraveling life processes across multiple scales. Imaging methods have considerably improved over the past decades and now allow to grasp the inner workings of proteins, organelles, cells, organs and whole organisms. Not only do they allow us to visualize these events in their most-relevant context but also to accurately quantify underlying biomechanical features and, so, provide essential information for their understanding. In this Commentary, we review a palette of imaging (and biophysical) methods that are available to the scientific community for elucidating a wide array of biological events. We cover the most-recent developments in intravital imaging, light-sheet microscopy, superresolution imaging, and correlative light and electron microscopy. In addition, we illustrate how these technologies have led to important insights in cell biology, from the molecular to the whole-organism resolution. Altogether, this review offers a snapshot of the current and state-of-the-art imaging methods that will contribute to the understanding of life and disease.

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X-Ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-Face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

Cited by 70 publications

References 23 publications

Contacts between the endoplasmic reticulum and other membranes in neurons

Contacts between the endoplasmic reticulum and other membranes in neurons

3‐D EM exploration of the hepatic microarchitecture ‐ lessons learned for large‐volume in situ serial sectioning

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology

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