scholarly journals Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

2019 ◽  
Author(s):  
Yury S. Bykov ◽  
Nir Cohen ◽  
Natalia Gabrielli ◽  
Hetty Manenschijn ◽  
Sonja Welsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Throughput ◽  
Cell Biology ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
Large Datasets ◽  
Genetic Screens ◽  
Resolution Limit ◽  
Computer Image Analysis ◽  
Multiple Cell

AbstractGenetic screens performed using high-throughput fluorescent microscopes have generated large datasets that have contributed many insights into cell biology. However, such approaches typically cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) is not subject to this resolution limit, generating detailed images of cellular ultrastructure, but requires time consuming preparation of individual samples, limiting its throughput. Here we overcome this obstacle and describe a robust method for screening by high-throughput electron microscopy. Our approach uses combinations of fluorophores as barcodes to mark the genotype of each cell in mixed populations, and correlative light and electron microscopy to read the fluorescent barcode of each cell before it is imaged by electron microscopy. Coupled with an easy-to-use software workflow for correlation, segmentation and computer image analysis, our method allows to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate the method on several organelles with samples that each contain up to 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher-throughput, and can be utilized in multiple ways to enable electron microscopy to become a powerful screening methodology.

scholarly journals High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding

2019 ◽  
Vol 218 (8) ◽  
pp. 2797-2811 ◽  
Author(s):  
Yury S. Bykov ◽  
Nir Cohen ◽  
Natalia Gabrielli ◽  
Hetty Manenschijn ◽  
Sonja Welsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Sample Preparation ◽  
High Throughput ◽  
Fluorescent Microscopy ◽  
Large Datasets ◽  
Genetic Screens ◽  
Cell Type ◽  
Resolution Limit ◽  
Computer Image Analysis ◽  
Multiple Cell

Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called “MultiCLEM,” allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique.

Simultaneous observations of immunolabeled frozen sections in lm and em

1978 ◽  
Vol 36 (2) ◽  
pp. 164-165
Author(s):  
K. T. Tokuyasu ◽  
J. Slot ◽  
S. J. Singer
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
Frozen Sections ◽  
Cover Glass ◽  
Rat Pancreas ◽  
Light Microscopic Observation ◽  
Rabbit Igg ◽  
Ultrathin Frozen Sections ◽  
Number Of Cells

Immunofluorescent microscopy is more suitable for the analysis of a large number of cells, often greater in the sensitivity for the detection of antigens, and more readily applicable for the identification of multiple antigens than immunoe1ectron microscopy. For combining these features of fluorescent microscopy with the superior resolution of electron microscopy, we attempted to observe the same immunolabeled ultrathin frozen sections with both light and electron microscopy.Ultrathin frozen sections of rat pancreas fixed in a mixture of 2% formaldehyde and 0.2% g1utaraldehyde for 1 hr at 4°C were first immunostained with rabbit anti-rat amylase antibodies, then very lightly with ferritin-goat anti-rabbit IgG conjugates and heavily with rhodamine-goat anti-rabbit IgG conjugates. For light microscopic observation, grids were suspended underneath the cover glass with a very thin layer of 50-90% glycerol and the cover glass was separated from the slide glass by a spacer to avoid the contact of the grid with the slide glass. After the light microscope observation, the grids were floated on 0.1 M phosphate buffer by dissolving glycerol into the buffer and processed for electron microscopy.

Cryo-fixation and associated developments in transmission electron microscopy: a cool future for nematology

Nematology ◽  
2016 ◽  
Vol 18 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Wim Bert ◽  
Dieter Slos ◽  
Olivier Leroux ◽  
Myriam Claeys
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Model Organisms ◽  
Fixation Method ◽  
Dimensional Reconstruction ◽  
Fixation Techniques

At present, the importance of sample preparation equipment for electron microscopy represents the driving force behind major breakthroughs in microscopy and cell biology. In this paper we present an introduction to the most commonly used cryo-fixation techniques, with special attention paid towards high-pressure freezing followed by freeze substitution. Techniques associated with cryo-fixation, such as immunolocalisation, cryo-sectioning, and correlative light and electron microscopy, are also highlighted. For studies that do not require high resolution, high quality results, or the immediate arrest of certain processes, conventional methods will provide answers to many questions. For some applications, such as immunocytochemistry, three-dimensional reconstruction of serial sections or electron tomography, improved preservation of the ultrastructure is required. This review of nematode cryo-fixation highlights that cryo-fixation not only results in a superior preservation of fine structural details, but also underlines the fact that some observations based on results solely obtained through conventional fixation approaches were either incorrect, or otherwise had severe limitations. Although the use of cryo-fixation has hitherto been largely restricted to model organisms, the advantages of cryo-fixation are sufficiently self-evident that we must conclude that the cryo-fixation method is highly likely to become the standard for nematode fixation in the near future.

A4Correlative Light and Electron Microscopy in Cell Biology

Microscopy ◽  
2015 ◽  
Vol 64 (suppl 1) ◽  
pp. i9.3-i9
Author(s):  
Céline Loussert Fonta ◽  
Caroline Kizilyaprak ◽  
Jean Daraspe ◽  
Willy Blanchard ◽  
Bruno M. Humbel
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Light And Electron Microscopy

scholarly journals Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Pieter Baatsen ◽  
Sergio Gabarre ◽  
Katlijn Vints ◽  
Rosanne Wouters ◽  
Dorien Vandael ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Fluorescent Proteins ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Fluorescence Signal ◽  
Microscopy Imaging ◽  
Tissue Samples ◽  
Rapid Acquisition

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

scholarly journals Three-dimensional nanostructure of an intact microglia cell

2018 ◽  
Author(s):  
Giulia Bolasco ◽  
Laetitia Weinhard ◽  
Tom Boissonnet ◽  
Ralph Neujahr ◽  
Cornelius T. Gross
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Microglia Cell ◽  
Cell Structures ◽  
Transmission Electron ◽  
Morphological Criteria ◽  
Membrane Protrusions ◽  
Microscopy Techniques

Microglia are non-neuronal cells of the myeloid lineage that invade and take up long-term residence in the brain during development (Ginhoux et al. 2010) and are increasingly implicated in neuronal maturation, homeostasis, and pathology (Bessis et al. 2007; Paolicelli et al. 2011; Li et al. 2012; Aguzzi et al. 2013, Cunningham 2013, Cunningham et al. 2013). Since the early twentieth century several methods for staining and visualizing microglia have been developed. Scientists in Ramón y Cajal’s group (Achúcarro 1913, Río-Hortega 1919) pioneered these methods and their work led to the christening of microglia as the third element of the nervous system, distinct from astrocytes and neurons. More recently, a combination of imaging, genetic, and immunological tools has been used to visualize microglia in living brain (Davalos et al. 2005; Nimmerjahn et al. 2005). It was found that microglia are highly motile under resting conditions and rapidly respond to injuries (Kettenmann et al. 2011) suggesting a role for microglia in both brain homeostasis and pathology. Transmission Electron microscopy (TEM) has provided crucial complementary information on microglia morphology and physiology but until recently EM analyses have been limited to single or limited serial section studies (Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012; Tremblay et al. 2012; Sipe et al. 2016). TEM studies were successful in defining a set of morphological criteria for microglia: a polygonal nucleus with peripheral condensed chromatin, a relatively small cytoplasm with abundant presence of rough endoplasmic reticulum (RER), and a large volume of lysosomes and inclusions in the perikaryon. Recent advances in volumetric electron microscopy techniques allow for 3D reconstruction of large samples at nanometer-resolution, thus opening up new avenues for the understanding of cell biology and architecture in intact tissues. At the same time, correlative light and electron microscopy (CLEM) techniques have been extended to 3D brain samples to help navigate and identify critical molecular landmarks within large EM volumes (Briggman and Denk 2006; Maco et al. 2013; Blazquez-Llorca et al. 2015, Bosch et al. 2015). Here we present the first volumetric ultrastructural reconstruction of an entire mouse hippocampal microglia using serial block face scanning electron microscopy (SBEM). Using CLEM we have ensured the inclusion of both large, small, and filopodial microglia processes. Segmentation of the dataset allowed us to carry out a comprehensive inventory of microglia cell structures, including vesicles, organelles, membrane protrusions, and processes. This study provides a reference that can serve as a data mining resource for investigating microglia cell biology.

scholarly journals Correlation of organelle dynamics between light microscopic live imaging and electron microscopic 3D architecture using FIB-SEM

Microscopy ◽  
2020 ◽  
Author(s):  
Keisuke Ohta ◽  
Shingo Hirashima ◽  
Yoshihiro Miyazono ◽  
Akinobu Togo ◽  
Kei-ichiro Nakamura
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Optical Microscope ◽  
Live Imaging ◽  
Reconstruction Technique ◽  
Electron Microscopic

Abstract Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

Localization of immunolabels by electron microscopy and image averaging

1983 ◽  
Vol 41 ◽  
pp. 282-283
Author(s):  
J. Frank ◽  
P.-Y. Sizaret ◽  
A. Verschoor ◽  
J. Lamy
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Attachment Site ◽  
Uranyl Acetate ◽  
Limulus Polyphemus ◽  
Resolution Limit ◽  
Electron Microscopic ◽  
Image Averaging ◽  
Top View ◽  
Better Than

The accuracy with which the attachment site of immunolabels bound to macromolecules may be localized in electron microscopic images can be considerably improved by using single particle averaging. The example studied in this work showed that the accuracy may be better than the resolution limit imposed by negative staining (∽2nm).The structure used for this demonstration was a halfmolecule of Limulus polyphemus (LP) hemocyanin, consisting of 24 subunits grouped into four hexamers. The top view of this structure was previously studied by image averaging and correspondence analysis. It was found to vary according to the flip or flop position of the molecule, and to the stain imbalance between diagonally opposed hexamers (“rocking effect”). These findings have recently been incorporated into a model of the full 8 × 6 molecule.LP hemocyanin contains eight different polypeptides, and antibodies specific for one, LP II, were used. Uranyl acetate was used as stain. A total of 58 molecule images (29 unlabelled, 29 labelled with antl-LPII Fab) showing the top view were digitized in the microdensitometer with a sampling distance of 50μ corresponding to 6.25nm.

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