Publication

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Kuipers, J., Kalicharan, R. D., Wolters, A. H. G., van Ham, T. J. & Giepmans, B. N. G., 25-May-2016, In : Journal of visualized experiments : JoVE. 111, 7 p., e53635.

Research output: Contribution to journalArticleAcademicpeer-review

APA

Kuipers, J., Kalicharan, R. D., Wolters, A. H. G., van Ham, T. J., & Giepmans, B. N. G. (2016). Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain. Journal of visualized experiments : JoVE, (111), [e53635]. https://doi.org/10.3791/53635

Author

Kuipers, Jeroen ; Kalicharan, Ruby D. ; Wolters, Anouk H G ; van Ham, Tjakko J ; Giepmans, Ben N G. / Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain. In: Journal of visualized experiments : JoVE. 2016 ; No. 111.

Harvard

Kuipers, J, Kalicharan, RD, Wolters, AHG, van Ham, TJ & Giepmans, BNG 2016, 'Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain', Journal of visualized experiments : JoVE, no. 111, e53635. https://doi.org/10.3791/53635

Standard

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain. / Kuipers, Jeroen; Kalicharan, Ruby D.; Wolters, Anouk H G; van Ham, Tjakko J; Giepmans, Ben N G.

In: Journal of visualized experiments : JoVE, No. 111, e53635, 25.05.2016.

Research output: Contribution to journalArticleAcademicpeer-review

Vancouver

Kuipers J, Kalicharan RD, Wolters AHG, van Ham TJ, Giepmans BNG. Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain. Journal of visualized experiments : JoVE. 2016 May 25;(111). e53635. https://doi.org/10.3791/53635


BibTeX

@article{97fbf0a6224b44839770a6ae92df23b1,
title = "Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain",
abstract = "Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically similar to 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.",
keywords = "Developmental Biology, Issue 111, Large-scale electron microscopy, scanning EM, nanotomy, zebrafish, quantitative EM, correlated microscopy, APOPTOTIC CELLS, RESOLUTION, IDENTIFICATION, ORGANELLES, MACROPHAGE, DYNAMICS, TISSUE, LIGHT, MAPS",
author = "Jeroen Kuipers and Kalicharan, {Ruby D.} and Wolters, {Anouk H G} and {van Ham}, {Tjakko J} and Giepmans, {Ben N G}",
year = "2016",
month = may,
day = "25",
doi = "10.3791/53635",
language = "English",
journal = "Journal of Visualized Experiments",
issn = "1940-087X",
publisher = "JOURNAL OF VISUALIZED EXPERIMENTS",
number = "111",

}

RIS

TY - JOUR

T1 - Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

AU - Kuipers, Jeroen

AU - Kalicharan, Ruby D.

AU - Wolters, Anouk H G

AU - van Ham, Tjakko J

AU - Giepmans, Ben N G

PY - 2016/5/25

Y1 - 2016/5/25

N2 - Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically similar to 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

AB - Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically similar to 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

KW - Developmental Biology

KW - Issue 111

KW - Large-scale electron microscopy

KW - scanning EM

KW - nanotomy

KW - zebrafish

KW - quantitative EM

KW - correlated microscopy

KW - APOPTOTIC CELLS

KW - RESOLUTION

KW - IDENTIFICATION

KW - ORGANELLES

KW - MACROPHAGE

KW - DYNAMICS

KW - TISSUE

KW - LIGHT

KW - MAPS

U2 - 10.3791/53635

DO - 10.3791/53635

M3 - Article

C2 - 27285162

JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

IS - 111

M1 - e53635

ER -

ID: 35514385