scholarly journals Automated vitrification of cryo-EM samples with controllable sample thickness using suction and real-time optical inspection

2021 ◽  
Author(s):  
Roman Koning ◽  
Hildo Vader ◽  
Martijn van Nugteren ◽  
Peter Grocutt ◽  
Wen Yang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
User Interaction ◽  
Lipid Vesicles ◽  
Dew Point ◽  
Particle Analysis ◽  
Specimen Preparation ◽  
Thin Film Preparation ◽  
And Performance

Abstract Speed and efficiency of data collection and image processing in cryo electron microscopy have increased over the last decade. However, cryo specimen preparation techniques have lagged behind and faster, more reproducible specimen preparation devices are needed. Here we present a new vitrification device with highly automated sample handling, requiring only limited user interaction. Moreover, the device allows inspection of thin films using light microscopy, since excess liquid is removed through suction by tubes, not blotting paper. In combination with dew-point control, this enables thin film preparation in a controlled and reproducible manner. The advantage is that quality of the prepared cryo specimen is characterized prior to electron microscopy data acquisition. Practicality and performance of the device are illustrated by experimental results obtained by vitrification of protein suspensions, lipid vesicles, bacterial and human cells, followed by imaged using single particle analysis, cryo electron tomography and cryo correlated light and electron microscopy.

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 A Survey of the Use of Iterative Reconstruction Algorithms in Electron Microscopy

2017 ◽  
Vol 2017 ◽  
pp. 1-17 ◽  
Author(s):  
C. O. S. Sorzano ◽  
J. Vargas ◽  
J. Otón ◽  
J. M. de la Rosa-Trevín ◽  
J. L. Vilas ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Tomographic Reconstruction ◽  
Three Dimensional ◽  
Iterative Algorithms ◽  
Projection Methods ◽  
Particle Analysis ◽  
Reconstruction Algorithms ◽  
Large Families ◽  
Key Steps

One of the key steps in Electron Microscopy is the tomographic reconstruction of a three-dimensional (3D) map of the specimen being studied from a set of two-dimensional (2D) projections acquired at the microscope. This tomographic reconstruction may be performed with different reconstruction algorithms that can be grouped into several large families: direct Fourier inversion methods, back-projection methods, Radon methods, or iterative algorithms. In this review, we focus on the latter family of algorithms, explaining the mathematical rationale behind the different algorithms in this family as they have been introduced in the field of Electron Microscopy. We cover their use in Single Particle Analysis (SPA) as well as in Electron Tomography (ET).

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.

Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies

2019 ◽  
Vol 21 (1) ◽  
pp. 395-415 ◽  
Author(s):  
Jana Ognjenović ◽  
Reinhard Grisshammer ◽  
Sriram Subramaniam
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Specimen Preparation ◽  
Macromolecular Assemblies ◽  
Cryo Electron Microscopy ◽  
G Protein Coupled ◽  
Improved Methods

In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein–coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

Low-Voltage, High-Resolution, Scanning Electron Microscopy of Platelet-Bound Fibrinogen

1996 ◽  
Vol 54 ◽  
pp. 316-317
Author(s):  
S.R. Simmons ◽  
R.M. Albrecht
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Low Voltage ◽  
Blood Clot ◽  
Light And Electron Microscopy ◽  
Essential Element ◽  
Specimen Preparation ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Adhesive Interactions

An essential element in blood clot formation is fibrinogen-mediated platelet aggregation. Fibrinogen is an adhesive plasma protein which binds to the αIIbp3 integrin on activated platelet surfaces. Platelets do not aggregate in the absence of fibrinogen binding, and fibrinogen bound to surfaces of platelets in aggregates is localized to regions of platelet-platelet contact. The fibrinogen molecule is symmetrical and bifunctional and may directly bridge the gap between platelets to bind to receptors on two adjacent platelets. However, the precise mechanism by which fibrinogen links platelets is unclear.Previously we have utilized colloidal gold labeling with correlative light and electron microscopy to investigate the binding of fibrinogen to receptors on surfaces of spread, substrate adherent platelets. The initial binding of gold-conjugated fibrinogen (FgnAu) and subsequent ligand-triggered receptor movement was followed on living platelets by video-enhanced light microscopy. Fibrinogen receptors initially are dispersed over much of the platelet surface and move centripetally upon fibrinogen binding, ultimately forming a band of bound fibrinogen on the platelet surface overlying a densely woven band of actin filaments surrounding the central granulomere. After preparation for electron microscopy, the same platelets as were followed in the light microscope were located in the high voltage TEM and the low voltage, high resolution, SEM (Hitachi S-900) and the final locations of the gold labeled receptor/ligand complexes were determined relative to internal or surface ultrastructure, respectively. More recently, we have utilized the SEM operated at low (1-2 kV) beam voltage to examine in detail the binding of unlabeled fibrinogen to platelets. With appropriate specimen preparation, individual cell surface macromolecules can be resolved in situ by low voltage SEM. In addition to the centripetal receptor redistribution seen with FgnAu, unlabeled fibrinogen appeared to undergo self-adhesive interactions following binding to platelet fibrinogen receptors, forming small, branched and globular protein aggregates during translocation across the platelet surface.(Fig. 1)

scholarly journals Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy

2019 ◽  
Vol 25 (4) ◽  
pp. 942-949 ◽  
Author(s):  
Lauren Ann Metskas ◽  
John A. G. Briggs
Keyword(s):  
Electron Microscopy ◽  
Membrane Fusion ◽  
Room Temperature ◽  
Light And Electron Microscopy ◽  
Lipid Vesicles ◽  
Morphological Description ◽  
Spectral Shifts ◽  
Cryo Electron Microscopy ◽  
State Duration ◽  
Fusion Applications

AbstractCorrelated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD, and its autoquenching by resonant energy transfer (RET), can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by RET can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.

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.

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