Correlated light and electron microscopy: ultrastructure lights up!

Boer, Pascal de; Hoogenboom, Jacob P.; Giepmans, Ben N. G.

doi:10.1038/nmeth.3400

Cited by 447 publications

(394 citation statements)

References 114 publications

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“…HTML-zoomable tissue EM files are very useful for quantification, sharing data that may not have been analyzed to its full content, combining LM and EM data (CLEM) 8 , presentation purposes in scientific research, to analyze patient data, and for education. Alternatively, in large-scale EM in a SEM, but not in a TEM, silica wafers can be used, which has two main advantages: BSD can be used, which is more generally available on scanning electron microscopes than STEM detection.…”

Section: Discussionmentioning

confidence: 99%

“…CLEM -In addition to facilitating quantitative EM, large scale EM makes it easier to correlate light microscopic labeling to EM level 8 . In the present example the presence of phagocytic microglia in a zebrafish ablation model is shown.…”

Section: Discussionmentioning

confidence: 99%

“…Advances include 3D EM, large scale 2D EM and improved methods and reagents for correlated light microscopy and electron microscopy (CLEM) to compare other modes of microscopic analysis directly to the EM level [8][9][10] . Large-scale 2D EM is particularly suitable to quantify or identify (novel) disease features for human pathology, study animal models for disease and tissue culture models.…”

Section: Introductionmentioning

confidence: 99%

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

Kuipers¹,

Kalicharan²,

Wolters³

et al. 2016

JoVE

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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][2][3][4][5][6][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 onehole 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 ~ 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][2][3][4][5] . Here, zebrafish brains were analyzed in a noninvasive 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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Kuipers¹,

Kalicharan²,

Wolters³

et al. 2016

JoVE

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“…We first took a correlative LM and EM approach (CLEM; de Boer et al, 2015) for the chloroplast samples. A frozenhydrated chloroplast appears green under cryo-LM, indicating the presence of chlorophylls (Fig.…”

Section: Structural Analysis Of Frozen-hydrated Biological Samplesmentioning

confidence: 99%

Cryogenic coherent X-ray diffraction imaging of biological samples at SACLA: a correlative approach with cryo-electron and light microscopy

Takayama

Yonekura

2016

Acta Cryst Sect A

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Coherent X-ray diffraction imaging at cryogenic temperature (cryo-CXDI) allows the analysis of internal structures of unstained, non-crystalline, whole biological samples in micrometre to sub-micrometre dimensions. Targets include cells and cell organelles. This approach involves preparing frozen-hydrated samples under controlled humidity, transferring the samples to a cryo-stage inside a vacuum chamber of a diffractometer, and then exposing the samples to coherent X-rays. Since 2012, cryo-coherent diffraction imaging (CDI) experiments have been carried out with the X-ray free-electron laser (XFEL) at the SPring-8 Å ngstrom Compact free-electron LAser (SACLA) facility in Japan. Complementary use of cryo-electron microscopy and/or light microscopy is highly beneficial for both pre-checking samples and studying the integrity or nature of the sample. This article reports the authors' experience in cryo-XFEL-CDI of biological cells and organelles at SACLA, and describes an attempt towards reliable and higher-resolution reconstructions, including signal enhancement with strong scatterers and Patterson-search phasing.

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“…However, a major drawback of these Cryo-electron tomography and subtomogram averaging technique is their limited resolution. Along with the technical innovation described above, further improvements, including focused ion beam micromachining to prepare samples with ideal thicknesses (Rigort et al, 2012;Villa et al, 2013), correlative light microscopyelectron microscopy (CLEM) (de Boer et al, 2015), and image processing algorithms such as denoising, template matching, and subtomogram averaging, as well as integration of complementary information on protein stoichiometry, proximity, and conformational variability with a highresolution structural repertoire contained in a database may enable Cryo-electron tomography to be developed as an unbiased discovery tool, namely in visual proteomics (Beck & Baumeister, 2016;Nickell et al, 2006).…”

mentioning

confidence: 99%

The Nobel Prize in Chemistry 2017: High-Resolution Cryo-Electron Microscopy

Chung

Kim

2017

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. They all contributed to the development of a Cryo-electron microscopy (EM) technique for determining the highresolution structures of biomolecules in solution, particularly without crystal and with much less amount of biomolecules than X-ray crystallography. In this brief commentary, we address the major advances made by these three Nobel laureates as well as the current status and future prospects of this Cryo-EM technique.

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Correlated light and electron microscopy: ultrastructure lights up!

Cited by 447 publications

References 114 publications

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

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

Cryogenic coherent X-ray diffraction imaging of biological samples at SACLA: a correlative approach with cryo-electron and light microscopy

The Nobel Prize in Chemistry 2017: High-Resolution Cryo-Electron Microscopy

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