scholarly journals Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
José María Mateos ◽  
Bruno Guhl ◽  
Jana Doehner ◽  
Gery Barmettler ◽  
Andres Kaech ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Super Resolution

scholarly journals An Easy Path for Correlative Electron and Super-Resolution Light Microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorothea Pinotsi ◽  
Simona Rodighiero ◽  
Silvia Campioni ◽  
Gabor Csucs
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light Microscopy ◽  
Amyloid Fibrils ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Wide Field ◽  
Set Up ◽  
Bio Compatibility ◽  
Scanning Electron

Abstract A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows. Here we present a way to overcome these difficulties by exploiting a commercially available wide-field fluorescence microscope integrated in the specimen chamber of a Scanning Electron Microscope (SEM) to perform correlative LM/EM studies. Super-resolution light microscopy was achieved by using a recently developed algorithm - the Super-Resolution Radial Fluctuations (SRRF) - to improve the resolution of diffraction limited fluorescent images. With this combination of hardware/software it is possible to obtain correlative super-resolution light and scanning electron microscopy images in an easy and fast way. The imaging workflow is described and demonstrated on fluorescently labelled amyloid fibrils, fibrillar protein aggregates linked to the onset of multiple neurodegenerative diseases, revealing information about their polymorphism.

Correlative STED super-resolution light and electron microscopy on resin sections

2019 ◽  
Vol 52 (37) ◽  
pp. 374003
Author(s):  
Christian A Wurm ◽  
Heinz Schwarz ◽  
Daniel C Jans ◽  
Dietmar Riedel ◽  
Bruno M Humbel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Super Resolution

scholarly journals Direct Visualization of Actin Filaments and Actin-Binding Proteins in Neuronal Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Minkyo Jung ◽  
Doory Kim ◽  
Ji Young Mun
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Actin Binding ◽  
Direct Visualization ◽  
Actin Binding Proteins ◽  
Synapse Plasticity

Actin networks and actin-binding proteins (ABPs) are most abundant in the cytoskeleton of neurons. The function of ABPs in neurons is nucleation of actin polymerization, polymerization or depolymerization regulation, bundling of actin through crosslinking or stabilization, cargo movement along actin filaments, and anchoring of actin to other cellular components. In axons, ABP–actin interaction forms a dynamic, deep actin network, which regulates axon extension, guidance, axon branches, and synaptic structures. In dendrites, actin and ABPs are related to filopodia attenuation, spine formation, and synapse plasticity. ABP phosphorylation or mutation changes ABP–actin binding, which regulates axon or dendritic plasticity. In addition, hyperactive ABPs might also be expressed as aggregates of abnormal proteins in neurodegeneration. Those changes cause many neurological disorders. Here, we will review direct visualization of ABP and actin using various electron microscopy (EM) techniques, super resolution microscopy (SRM), and correlative light and electron microscopy (CLEM) with discussion of important ABPs in neuron.

scholarly journals Innenarchitektur der Zellkraftwerke — Membranfalten in Hochauflösung

BIOspektrum ◽  
2021 ◽  
Vol 27 (2) ◽  
pp. 161-164
Author(s):  
Till Stephan ◽  
Peter Ilgen ◽  
Stefan Jakobs
Keyword(s):  
Electron Microscopy ◽  
Protein Complex ◽  
Mitochondrial Protein ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Eukaryotic Cells ◽  
Inner Membrane ◽  
Double Membrane ◽  
Cellular Organelles

AbstractMitochondria are essential cellular organelles, which supply eukaryotic cells with the universal energy carrier adenosine triphosphate. These organelles feature a unique double-membrane architecture, which is formed by a smooth outer membrane and a highly folded inner membrane. Harnessing super-resolution light and electron microscopy, we investigate the role of MICOS, a large mitochondrial protein complex, in determining the complex folding of the inner membrane.

scholarly journals Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maarten W. Tuijtel ◽  
Abraham J. Koster ◽  
Stefan Jakobs ◽  
Frank G. A. Faas ◽  
Thomas H. Sharp
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution

scholarly journals Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Simon Peters ◽  
Lena Kaiser ◽  
Julian Fink ◽  
Fabian Schumacher ◽  
Veronika Perschin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Antimicrobial Activity ◽  
Outer Membrane ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Hydroxyl Group ◽  
Side Chain ◽  
Gram Negative ◽  
Array Tomography ◽  
Transmission Electron

AbstractSphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.

scholarly journals A New Solution of Non-integrated Correlative Light and Electron Microscopy Based on High-vacuum Optical Platform

2016 ◽  
Vol 22 (S3) ◽  
pp. 248-249
Author(s):  
Shuoguo Li ◽  
Gang Ji ◽  
Xiaojun Huang ◽  
Lei Sun ◽  
Jianguo Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Vacuum ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Fluorescent Imaging ◽  
Three Dimensions ◽  
Large Field ◽  
Correlative Imaging ◽  
Structural Preservation ◽  
High Level

Abstract Correlative light and electron microscopy (CLEM) offers a means of guiding the search for the unique or rare events by fluorescence microscopy (FM) and allows electron microscopy (EM) to zoom in on them for subsequent EM examination in three-dimensions (3D) and with nanometer-scale resolution. FM visualizes the localization of specific antigens by using fluorescent tags or proteins in a large field-of-view to study their cellular function, whereas EM provides the high level of resolution for complex structures. And cryo CLEM combines the advantages of maintaining structural preservation in a near-native state throughout the entire imaging process and by avoiding potentially harmful pre-treatments, such as chemical fixation, dehydration and staining with heavy metals. Besides for frozen-hydrated biological samples, CLEM combines the advantages of a close-to-life preservation of biological materials by keeping them embedded in vitreous ice throughout the entire imaging process and the frozen-hydrated condition is very suitable to maintain fluorescent signals. In recent years, many new instruments and software which intended to optimize the workflow and to obtain better experimental results of CLEM have been presented or even commoditized. While, the specimen damage during transfer from FM to EM and the resolution of CLEM were still need to be improved. Here we set up a High-vacuum Optical Platform to develop CLEM imaging technology (HOPE), which was designed to realize high-vacuum optical ( fluorescent) imaging for cryo-sample on EM cryo-holder (e.g. Gatan 626). A non-integrated high-vacuum cryo-optical stage, which adapted to the EM cryo holder, was fixed on epi-fluorescence microscope (or super-resolution microscope) to obtain fluorescent images. And then the EM cryo holder would be transferred to EM for collection of EM data. This protocol was aimed to minimize the specimen damage during transfer from FM to EM and it was versatile to expend to different types of light microscopy or electron microscopy. Our HOPE had already passed correlative imaging test, and the results showed that it was convenient and effective.

scholarly journals Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale

2021 ◽  
Vol 72 (1) ◽  
Author(s):  
Peter D. Dahlberg ◽  
W.E. Moerner
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Nanometer Scale ◽  
Annual Review ◽  
Publication Date ◽  
Fluorescence And Electron Microscopy ◽  
Cellular Context

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 72 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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