A simple preparation method for CLEM using pre-embedding immunohistochemistry with a novel fluorescent probe and stable embedding resin

Microscopy ◽  
2021 ◽  
Author(s):  
Takaaki Kanemaru ◽  
Teruyoshi Kondo ◽  
Kei-ichiro Nakamura ◽  
Hiroyuki Morimoto ◽  
Kentaro Nishi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
High Resolution ◽  
Light Microscopy ◽  
Fluorescent Probe ◽  
Region Of Interest ◽  
Wide Field ◽  
Lr White ◽  
Fluorescence Light Microscopy ◽  
Fluorescence Light

Abstract Correlative light and electron microscopy (CLEM) is an excellent approach for examining the cellular localization of biomolecules. Here, we developed a simple method for CLEM by combining pre-embedding immunohistochemistry with a novel fluorescent probe, namely Fluolid NS Orange, and an embedding resin called ʻDurcupan™ʼ. Specimens were embedded in Durcupan™ or LR White after immunolabeling and post-fixation using glutaraldehyde and osmium tetroxide. Next, ultrathin sections were prepared on a finder grid with navigation markers. The section of the specimen embedded in Durcupan™ was found to be more stable against electron beam irradiation than specimens embedded in LR White. A fluorescence light microscopy image and a transmission electron microscopy (TEM) image, at wide-field, and low magnification, were independently obtained with the same ultrathin section. Using the three corners between finder grid bars as landmarks, fluorescence light microscopy images were superimposed with wide-field, low-magnification TEM images to identify the region of interest, which was subsequently enlarged to ascertain cellular structures localized beneath fluorescent signals. However, the enlarged TEM images appeared blurred, and fluorescence signals had a hazy appearance. To resolve this, the enlarged TEM images were replaced by high-resolution TEM images focused directly on the region of interest, thereby facilitating the collection of high-resolution CLEM images. The simple sample processing method for CLEM using osmium-resistant Fluolid NS Orange and electron beam damage-resistant Durcupan™ allowed the determination of the precise localization of fluorescence signals at subcellular levels.

Advances in Correlative Single-Molecule Localization Microscopy and Electron Microscopy

2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Ulrike Endesfelder
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Single Molecule ◽  
Light And Electron Microscopy ◽  
Fluorescence Labeling ◽  
Localization Microscopy ◽  
Fluorescence Light Microscopy ◽  
Fluorescence Light ◽  
And Function ◽  
Single Molecule Localization Microscopy

AbstractDuring the last few decades, correlative fluorescence light and electron microscopy (FLM-EM) has gained increased interest in the life sciences concomitant with the advent of fluorescence light microscopy. It has become, accompanied by numerous developments in both techniques, an important tool to study bio-cellular structure and function as it combines the specificity of fluorescence labeling with the high structural resolution and cellular context information given by the EM images. Having the recently introduced single-molecule localization microscopy techniques (SMLM) at hand, FLM-EM can now make use of improved fluorescence light microscopy resolution, single-molecule sensitivity and quantification strategies. Here, currently used methods for correlative SMLM and EM including the special requirements in sample preparation and imaging routines are summarized and an outlook on remaining challenges concerning methods and instrumentation is provided.

Examination of Schistosome Cercariae and Schistosomules by Scanning Electron Microscopy

1969 ◽  
Vol 27 ◽  
pp. 50-51
Author(s):  
D. Johnson ◽  
P. Moriearty
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Light Microscopy ◽  
Surface Structure ◽  
Extensive Study ◽  
Scanning Microscopy ◽  
Host Parasite ◽  
Conventional Electron Microscopy ◽  
Scanning Electron

Since several species of Schistosoma, or blood fluke, parasitize man, these trematodes have been subjected to extensive study. Light microscopy and conventional electron microscopy have yielded much information about the morphology of the various stages; however, scanning electron microscopy has been little utilized for this purpose. As the figures demonstrate, scanning microscopy is particularly helpful in studying at high resolution characteristics of surface structure, which are important in determining host-parasite relationships.

High Resolution Scanning Electron Microscopy

1991 ◽  
Vol 49 ◽  
pp. 478-479
Author(s):  
David Joy ◽  
James Pawley
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Beam ◽  
Electron Microscope ◽  
High Resolution ◽  
Scanning Electron Microscope ◽  
Electron Probe ◽  
Impact Point ◽  
Interaction Volume ◽  
Scanning Electron

The scanning electron microscope (SEM) builds up an image by sampling contiguous sub-volumes near the surface of the specimen. A fine electron beam selectively excites each sub-volume and then the intensity of some resulting signal is measured. The spatial resolution of images made using such a process is limited by at least three factors. Two of these determine the size of the interaction volume: the size of the electron probe and the extent to which detectable signal is excited from locations remote from the beam impact point. A third limitation emerges from the fact that the probing beam is composed of a finite number of discrete particles and therefore that the accuracy with which any detectable signal can be measured is limited by Poisson statistics applied to this number (or to the number of events actually detected if this is smaller).

Quantitative high-resolution electron microscopy (HREM) of defects in ordered polymers

1996 ◽  
Vol 54 ◽  
pp. 166-167
Author(s):  
Patricia M. Wilson ◽  
David C. Martin
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
High Resolution ◽  
Liquid Crystalline ◽  
High Resolution Electron Microscopy ◽  
Polymer Materials ◽  
Scattered Electron ◽  
Crystalline Polymers ◽  
Resolution Electron ◽  
Beam Damage

Efforts in our laboratory and elsewhere have established the utility of low dose high resolution electron microscopy (HREM) for imaging the microstructure of crystalline and liquid crystalline polymers. In a number of polymer systems, direct imaging of the lattice spacings by HREM has provided information about the size, shape, and relative orientation of ordered domains in these materials. However, because of the extent of disorder typical in many polymer microstructures, and because of the sensitivity of most polymer materials to electron beam damage, there have been few studies where the contrast observed in HREM images has been analyzed in a quantitative fashion.Here, we discuss two instances where quantitative information about HREM images has been used to provide new insight about the organization of crystalline polymers in the solid-state. In the first, we study the distortion of the polymer lattice planes near the core of an edge dislocation and compare these results to theories of dislocations in anisotropic and liquid crystalline solids. In the second, we investigate the variations in HREM contrast near the edge of wedge-shaped samples. The polymer used in this study was the diacetylene DCHD, which is stable to electron beam damage (Jc = 20 C/cm2) and highly crystalline. The instrument used in this work was a JEOL 4000 EX HRTEM with a beam blanidng device. More recently, the 4000 EX has been installed with instrumentation for dynamically recording scattered electron beam currents.

High-Resolution Transmission Electron Microscopy of Initial Stage of Radiation Damage of YBa2Cu4OyInduced by 1 MV Electron Beam

1991 ◽  
Vol 30 (Part 2, No. 8A) ◽  
pp. L1375-L1377 ◽  
Author(s):  
Yoshio Matsui ◽  
Kasumi Yanagisawa ◽  
Naohito Fujiwara
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Electron Beam ◽  
High Resolution ◽  
Radiation Damage ◽  
Transmission Electron ◽  
Initial Stage

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.

scholarly journals High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy

Microscopy ◽  
2015 ◽  
Vol 64 (6) ◽  
pp. 387-394 ◽  
Author(s):  
Daisuke Koga ◽  
Satoshi Kusumi ◽  
Ryusuke Shodo ◽  
Yukari Dan ◽  
Tatsuo Ushiki
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Light Microscopy ◽  
High Resolution Imaging ◽  
Semithin Sections ◽  
Resolution Imaging ◽  
Scanning Electron

scholarly journals Analysis of the Performance of an 8k x 8k Lens Coupled Detector for Wide-field, High-resolution Transmission Electron Microscopy (TEM)

2005 ◽  
Vol 11 (S02) ◽  
Author(s):  
J C Bouwer ◽  
S T Peltier ◽  
L Jin ◽  
K Khodjasaryan ◽  
S Geist ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Wide Field ◽  
Transmission Electron
Sign in / Sign up

Export Citation Format

Share Document