Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Mukherjee, Konark; Clark, Helen R.; Chavan, Vrushali; Benson, Emily; Kidd, Grahame J.; Srivastava, Sarika

doi:10.3791/54214

Cited by 25 publications

(28 citation statements)

References 51 publications

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“…Tissues for SBF-SEM were fixed as above in modified Karnovsky fixative and stained and imaged by Renovo Neural Inc. Briefly, the samples were washed, stained with 1% tannic acid for 30 min and then incubated successively with osmium ferricyanide, tetracarbohydrazide, osmium tetroxide, uranyl acetate, and lead aspartate (67,68). Tissues were dehydrated and embedded in Epon-812 resin (Procure; Electron Microscopy Sciences).…”

Section: Sbf-em Imaging and Analysis Of Spinal Cord Sections From Catmentioning

confidence: 99%

The adult oligodendrocyte can participate in remyelination

Duncan

Radcliff

Heidari

et al. 2018

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

195

155

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Endogenous remyelination of the CNS can be robust and restore function, yet in multiple sclerosis it becomes less complete with time. Promoting remyelination is a major therapeutic goal, both to restore function and to protect axons from degeneration. Remyelination is thought to depend on oligodendrocyte progenitor cells, giving rise to nascent remyelinating oligodendrocytes. Surviving, mature oligodendrocytes are largely regarded as being uninvolved. We have examined this question using two large animal models. In the first model, there is extensive demyelination and remyelination of the CNS, yet oligodendrocytes survive, and in recovered animals there is a mix of remyelinated axons interspersed between mature, thick myelin sheaths. Using 2D and 3D light and electron microscopy, we show that many oligodendrocytes are connected to mature and remyelinated myelin sheaths, which we conclude are cells that have reextended processes to contact demyelinated axons while maintaining mature myelin internodes. In the second model in vitamin B 12 -deficient nonhuman primates, we demonstrate that surviving mature oligodendrocytes extend processes and ensheath demyelinated axons. These data indicate that mature oligodendrocytes can participate in remyelination.adult oligodendrocyte | remyelination | large animal models

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Section: Sbf-em Imaging and Analysis Of Spinal Cord Sections From Catmentioning

confidence: 99%

The adult oligodendrocyte can participate in remyelination

Duncan

Radcliff

Heidari

et al. 2018

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

195

155

View full text Add to dashboard Cite

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“…Because the ultrastructural features analyzed are at the nanometer scale, samples used for EM are typically very small (< 1mm 3 ), which facilitates proper sample preparation. In preparing samples, portions of the heart are typically excised, carefully prepared for imaging (fixed and stained) 14,38 , and then imaged with an EM modality 14,31 . Using this procedure, however, finding the ultrastructure associated with a specific microscopic feature can be daunting.…”

Section: Discussionmentioning

confidence: 99%

“…In order to enable both whole-heart micro-CT imaging and subsequent SBF-SEM imaging of regions of interest (ROIs), we processed fixed hearts for micro-CT using the initial portion of a Renovo Neural, Inc (Cleveland, USA) protocol 38 designed for SBF-SEM imaging (see Figure 1, Step 1 ). Each heart was placed in a 5 mL glass scintillation vial and we used 3 mL of solution per vial for each incubation/wash.…”

Section: Methodsmentioning

confidence: 99%

Multiscale cardiac imaging to capture the whole heart and its internal cellular architecture, with applications to congenital heart disease

Rykiel

López

McQuiston

et al. 2020

Preprint

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9Efficient cardiac pumping depends on the morphological structure of the heart, but also on its 10 sub-cellular (ultrastructural) architecture, which enables cardiac contraction. In cases of 11 congenital heart defects, localized sub-cellular disruptions in architecture that increase the risk 12 of heart failure are only starting to be discovered. This is in part due to a lack of technologies 13 that can image the three dimensional (3D) heart structure, assessing malformations; and its 14 ultrastructure, assessing disruptions. We present here a multiscale, correlative imaging 15 procedure that achieves high-resolution images of the whole heart, using 3D micro-computed 16 tomography (micro-CT); and its ultrastructure, using 3D scanning electron microscopy (SEM). 17This combination of technologies has not been possible before in imaging the same cardiac 18 sample due to the heart large size, even when studying small fetal and neonatal animal models 19 (~5x5x5mm 3 ). Here, we achieved uniform fixation and staining of the whole heart, without losing 20 ultrastructural preservation (at the nm resolution range). Our approach enables multiscale 21 studies of cardiac architecture in models of congenital heart disease and beyond. 22 23 51 whole heart morphology and its sub-cellular organization (ultrastructural architecture). Our 52 multiscale procedure uses micro computed tomography (micro-CT) imaging to capture heart 53 morphology at micrometer resolution, and scanning electron microscopy (SEM) to capture 54 cardiac tissue ultrastructure at nanometer resolution. Current SEM technologies allow for three-55 dimensional (3D) imaging of sub-cellular architecture, enabling reconstruction and quantification 56 of ultrastructural features within a tissue volume 14-16 . Among 3D SEM methods, we have 57 selected serial block-face SEM (SBF-SEM) for ultrastructural imaging, as it allows 3D imaging of 58 relatively large volumes (sample size 40x60x40 µm 3 ). The methodology we present herein 59 improves upon previous protocols by achieving uniform staining of a relatively large heart 60 sample (3-4 mm wide, 5-6 mm long), circumventing micro-CT x-ray penetration issues, and 61 allowing sample screening and selection prior to full sample preparation. Our multiscale imaging, 62 further, enables mapping of structural and ultrastructural heart features. 63 129 thickening and enlargement of the RV wall. RV hypertrophy in TOF, however, develops over 130 time as the stenosis of the pulmonary artery increases pressure in the RV after birth 17 and was 131 not present in the heart examined in this study (see Figure 3 for a comparison of the selected 132 normal and TOF hearts). The TOF heart analyzed here featured supravalvular pulmonary 133 stenosis, a ventricular septal defect, and an overriding aorta. The right ventricle was enlarged 134and thin-walled compared to the control heart (Figure 3). Further, the TOF heart was missing 135 the right branch of the pulmonary artery. In humans, this rare condition, called unilateral 136

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“…Also, and if available, having the ability to evaluate the use of FIB-SEM versus SBF-SEM will help the researcher to design a data collection strategy. The protocols evaluated during development of this workflow included the Dresden protocol (Paridaen, Wilsch-Bräuninger, & Huttner, 2013), Renovo (Mukherjee et al, 2016) and the Hua method (Hua, Laserstein, & Helmstaedter, 2015). We settled on the the Hua method with some modifications as described below, for human cancer biopsies.…”

Section: Methodsmentioning

confidence: 99%

A workflow for visualizing human cancer biopsies using large-format electron microscopy

Riesterer

López

Stempinski

et al. 2019

Preprint

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Recent developments in large format electron microscopy have enabled generation of images that provide detailed ultrastructural information on normal and diseased cells and tissues. Analyses of these images increase our understanding of cellular organization and interactions and disease-related changes therein. In this manuscript, we describe a workflow for two-dimensional (2D) and three-dimensional (3D) imaging, including both optical and scanning electron microscopy (SEM) methods, that allow pathologists and cancer biology researchers to identify areas of interest from human cancer biopsies. The protocols and mounting strategies described in this workflow are compatible with 2D large format EM 2 mapping, 3D focused ion beam-SEM and serial block face-SEM. The flexibility to use diverse imaging technologies available at most academic institutions makes this workflow useful and applicable for most life science samples. Volumetric analysis of the biopsies studied here revealed morphological, organizational and ultrastructural aspects of the tumor cells and surrounding environment that cannot be revealed by conventional 2D EM imaging. Our results indicate that although 2D EM is still an important tool in many areas of diagnostic pathology, 3D images of ultrastructural relationships between both normal and cancerous cells, in combination with their extracellular matrix, enables cancer researchers and pathologists to better understand the progression of the disease and identify potential therapeutic targets.

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Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Cited by 25 publications

References 51 publications

The adult oligodendrocyte can participate in remyelination

The adult oligodendrocyte can participate in remyelination

Multiscale cardiac imaging to capture the whole heart and its internal cellular architecture, with applications to congenital heart disease

A workflow for visualizing human cancer biopsies using large-format electron microscopy

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