Seeing the Forest for the Trees: Selective Staining and Contrast Enhancement Methods for Biological Electron Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 766-767
Author(s):  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Fluorescent Compound ◽  
Selective Staining ◽  
3 Dimensional ◽  
Cellular Processes ◽  
Wide Range ◽  
Invaluable Tool ◽  
Versatile Technique

Electron tomography has proven to be an invaluable tool for studying the 3-dimensional organization of a wide range of structures, from large cellular processes down to individual macromolecular complexes. An important requirement for electron tomography of thick specimens is the need for selective staining to delineate the structure of interest from other cellular constituents. of particular usefulness in this regard is the method of fluorescence photooxidation, whereby the reactive oxygen generated by a fluorescent compound is used to oxidize diaminobenzidine into a reaction product that can then be visualized with the electron microscope. The main advantages of this method are that not only does it allow for excellent correlated light and electron microscopy, but also the relatively small size of the oxidizing agent used allows for excellent 3-D labeling with high resolution.This method has proven to be a particularly versatile technique.

scholarly journals Multiscale Electron Microscopy for the Study of Viral Replication Organelles

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 197
Author(s):  
Georg Wolff ◽  
Montserrat Bárcena
Keyword(s):  
Electron Microscopy ◽  
Viral Replication ◽  
Electron Tomography ◽  
Viral Rna ◽  
Human Pathogens ◽  
Wide Range ◽  
Recent Developments ◽  
Versatile Technique ◽  
Positive Strand Rna

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.

scholarly journals A modular platform for automated cryo-FIB workflows

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sven Klumpe ◽  
Herman K H Fung ◽  
Sara K Goetz ◽  
Ievgeniia Zagoriy ◽  
Bernhard Hampoelz ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Integrity ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Software Tool ◽  
3 Dimensional ◽  
Improved Stability ◽  
Modular Platform ◽  
Beam Currents

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

Correlated 3D Light and Electron Microscopy of Large, Complex Structures: Analysis of Transverse Tubules in Heart Failure

1998 ◽  
Vol 4 (S2) ◽  
pp. 440-441
Author(s):  
Maryann E. Martone ◽  
Andrea Thor ◽  
Stephen J. Young ◽  
Mark H. Ellisman.
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Structural Features ◽  
Electron Microscopic Level ◽  
Specimen Preparation ◽  
Large Sample Size ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

Light microscopic imaging has experienced a renaissance in the past decade or so, as new techniques for high resolution 3D light microscopy have become readily available. Light microscopic (LM) analysis of cellular details is desirable in many cases because of the flexibility of staining protocols, the ease of specimen preparation and the relatively large sample size that can be obtained compared to electron microscopic (EM) analysis. Despite these advantages, many light microscopic investigations require additional analysis at the electron microscopic level to resolve fine structural features.High voltage electron microscopy allows the use of relatively thick sections compared to conventional EM and provides the basis for excellent new methods to bridge the gap between microanatomical details revealed by LM and EM methods. When combined with electron tomography, investigators can derive accurate 3D data from these thicker specimens. Through the use of correlated light and electron microscopy, 3D reconstructions of large cellular or subcellular structures can be obtained with the confocal microscope,

scholarly journals An Economical, Portable Manual Cryogenic Plunge Freezer for the Preparation of Vitrified Biological Samples for Cryogenic Electron Microscopy

2020 ◽  
Vol 26 (3) ◽  
pp. 413-418
Author(s):  
Jamie S. Depelteau ◽  
Gert Koning ◽  
Wen Yang ◽  
Ariane Briegel
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Bacterial Cells ◽  
Cellular Processes ◽  
Cryo Electron Tomography ◽  
Specialized Equipment ◽  
Improved Design ◽  
Commercial Equipment ◽  
Local Machine

AbstractVisualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

scholarly journals AutoCLEM: An Automated Workflow for Correlative Live-Cell Fluorescence Microscopy and Cryo-Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Xiaofeng Fu ◽  
Jiying Ning ◽  
Zhou Zhong ◽  
Zandrea Ambrose ◽  
Simon Charles Watkins ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Speed ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Live Cell ◽  
Host Cells ◽  
Spatiotemporal Information ◽  
Fluorescence Light ◽  
Correlation Process ◽  
Electron Imaging

AbstractCorrelative light and electron microscopy (CLEM) combines the strengths of both light and electron imaging modalities and enables linking of biological spatiotemporal information from live-cell fluorescence light microscopy (fLM) to high-resolution cellular ultra-structures from cryo-electron microscopy and tomography (cryoEM/ET). This has been previously achieved by using fLM signals to localize the regions of interest under cryogenic conditions. The correlation process, however, is often tedious and time-consuming with low throughput and limited accuracy, because multiple correlation steps at different length scales are largely carried out manually. Here, we present an experimental workflow, AutoCLEM, which overcomes the existing limitations and improves the performance and throughput of CLEM methods, and associated software. The AutoCLEM system encompasses a high-speed confocal live-cell imaging module to acquire an automated fLM grid atlas that is linked to the cryoEM grid atlas, followed by cryofLM imaging after freezing. The fLM coordinates of the targeted areas are automatically converted to cryoEM/ET and refined using fluorescent fiducial beads. This AutoCLEM workflow significantly accelerates the correlation efficiency between live-cell fluorescence imaging and cryoEM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells.

Melt electro written three-dimensional scaffolds engineered as oral microcosm models-an in vitro study.

2020 ◽  
Author(s):  
Srinivas Ramachandra ◽  
Abdulla Abdal-hay ◽  
Pingping Han ◽  
Ryan Lee ◽  
Saso Ivanovski
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Confocal Microscopy ◽  
Pore Size ◽  
Next Generation ◽  
16S Sequencing ◽  
3 Dimensional ◽  
Wide Range ◽  
Scanning Electron ◽  
Medical Grade

<p><strong>Introduction</strong>: Biofilms are 3-dimensional (3D) aggregates of microorganisms that are associated with a wide range of diseases. Although there have been several studies investigating biofilm formation on two-dimensional substrates, the use of 3D substrates may result in more representative and clinically relevant models. Accordingly, the aim of this study was to compare the growth of biofilms in the 3D substrates against biofilms grown in 2D substrates.<br /><strong>Material and Methods:</strong> Two grams of medical grade polycaprolactone (PCL) were loaded into a plastic Luer-lock 3 ml syringe and a 23G needle was used as a spinneret. The syringe was placed in a melt electro-writing (MEW) device to obtain fine fibers under controlled parameters. The 3-dimensional MEW PCL scaffolds were manufactured and characterised with an overall thickness of ~ 0.8 mm, with ~ 15 μm diameter fibers and ordered pore sizes of either 100 or 250 µm. PCL films employed as 2D substrates were manufactured by dissolving 10 gms of PCL in 100 ml chloroform and stirred for 3 h to obtain a transparent solution. Then, the solution was cast in glass petri dishes and dried to remove all organic solvents. In addition, commercial hydroxyapatite discs were also used as 2D controls. Unstimulated saliva from six healthy donors (gingival health) were used to grow biofilms. The formed biofilms were assessed at day 4, day 7 and day 10 using crystal violet assay, confocal microscopy, scanning electron microscopy and next-generation 16s sequencing.<br /><strong>Results:</strong> The results demonstrates that 3D PCL scaffolds dramatically enhanced biofilm biomass and thickness growth compared to that of the 2D controls. Confocal microscopy of biofilms at day 4 stained with SYTO 9 and propidium iodide showed thickness of biofilms in 2D substrates were 39 µm and 81µm for hydroxyapatite discs and PCL films, respectively. Biofilms in 3D substrates were 250 µm and 338 µm for MEW PCL 100µm pore size and MEW PCL 250 µm pore size, respectively. Similar results were noticed at day 7 and day 10. Scanning electron microscopy showed biofilm bridges formed over the fibers of the MEW scaffolds. Pilot trials of next generation sequencing detected similar taxa in biofilms formed in 3D scaffolds compared to that of 2D substrates.<br /><strong>Discussion:</strong> We have successfully investigated a 3D biofilm growth model using 3D medical grade PCL scaffolds. Thicker biofilms can be conveniently grown using this inexpensive static model. This will facilitate 3D microbial community studies that are more clinically relevant and improve our understanding of biofilm-associated disease processes.</p> <p> </p>

A simple technique for selective staining of neurosecretory products in epoxy sections with paraldehyde fuchsin

1977 ◽  
Vol 55 (9) ◽  
pp. 1571-1575 ◽  
Author(s):  
Colin G. H. Steel ◽  
G. P. Morris
Keyword(s):  
Electron Microscopy ◽  
Peracetic Acid ◽  
Simple Technique ◽  
Light And Electron Microscopy ◽  
Sodium Methylate ◽  
Neurosecretory Cells ◽  
Neurosecretory Material ◽  
Neurosecretory Granules ◽  
Thin Sections ◽  
Selective Staining

A technique for correlative light and electron microscopy of neurosecretory cells (NSC) is described. Neurosecretory material is selectively stained in 0.5-μm epoxy sections with paraldehyde fuchsin (PAF) after partial resin removal with sodium methylate in methanol and oxidation in peracetic acid. Adjacent 'thin' sections permit electron microscopy of the same cells. The organelles responsible for the affinity of NSC for PAF are in part neurosecretory granules and in part lysosomes. The technique reveals great ultrastructural differences between NSC which appear similar in the light microscope.

scholarly journals Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Steffen Klein ◽  
Benedikt H. Wimmer ◽  
Sophie L. Winter ◽  
Androniki Kolovou ◽  
Vibor Laketa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Current Knowledge ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
A549 Cells ◽  
Airway Epithelial Cells ◽  
Lamellar Bodies ◽  
Membrane Structures ◽  
Alveolar Cells ◽  
Cryo Electron Tomography

AbstractLamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through “T”-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.

scholarly journals Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes

2021 ◽  
Author(s):  
Sergey Loginov ◽  
Job Fermie ◽  
Jantina Fokkema ◽  
Alexandra V Agronskaia ◽  
Cecilia de Heus ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Temporal Organization ◽  
Data Sets ◽  
3 Dimensional ◽  
Cellular Physiology ◽  
3D Fluorescence Microscopy

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Directly linking molecular to nanoscale ultrastructural information is therefore crucial to understand cellular physiology. Volume or 3-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. Application of volume-CLEM is however hampered by limitations in throughput and 3D correlation efficiency. Addressing these limitations, we here describe a novel pipeline for volume-CLEM that provides high-precision (<100nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) data sets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated in a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume-EM, and obviates the need for post correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D.

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