低温电子显微技术在膜蛋白结构研究中的应用和展望

介绍了蛋白质电子晶体学和单颗粒分析技术这两种低温电子显微技术在膜蛋白和膜蛋白复合体结构研究中的具体方法和近10~20年来的实际应用,并分别分析了这两种方法的优势和瓶颈。此外,还介绍了Amphipol替代、Streptavidin二维晶体锚定脂质体和纳米球包被脂质体等近两年来出现的新的用于低温电镜成像的膜蛋白样品制备方法。最后对膜蛋白的低温电子显微研究的未来发展做了展望。     

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron‐microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high‐pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non‐correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron‐microscopic protocols, making them more accessible to a wider range of researchers. Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time‐demanding procedure of specimen preparation for any kind of TEM.     

Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

冷冻聚焦离子束-扫描电镜成像技术研究进展

冷冻聚焦离子束-扫描电镜成像(Cryo-FIB-SEM)是一种新兴的成像检测技术,在原位进行冷冻聚焦离子束切割和冷冻扫描电镜成像,为研究天然含水状态下生物样品内部未被破坏的原始结构打开了一扇窗口。近年来,该技术在生命科学领域的应用研究取得了一系列重要进展。该文对其在冷冻体积连续成像、冷冻光电关联成像、冷冻透射扫描成像、冷冻含水切片制备监控及冷冻扫描图像处理等方面的研究进展进行综述,并展望了该技术在大体积生物样品三维原位成像研究领域的前沿性发展趋势,以期推动Cryo-FIB-SEM技术在生物样品三维结构研究中的应用。     

Immobilization of biotinylated DNA on 2-D streptavidin crystals

The structural study of transient nucleoprotein complexes by electron microscopy is hampered by the coexistence of multiple interaction states leading to an heterogeneous image population. To tackle this problem, we have investigated the controlled immobilization of double stranded DNA molecules and of nucleoprotein complexes onto a support suitable for cryo-electron microscopy observation. The DNA was end-labeled with a biotin moiety in order to decorate, or to be incorporated into, two-dimensional streptavidin crystals formed in contact of a biotinylated lipid layer. The binding specificity and efficiency were examined by radioactively labeled oligonucleotides and by direct visualization of unstained and hydrated nucleic acid molecules in cryo-electron microscopy. By using RNA polymerase we further show that, once immobilized, femtomolar amounts of DNA template are suitable to interact with the enzyme. The image analysis of the RNA polymerase-DNA complexes showed that a three-dimensional model can be retrieved from such samples.     

Towards native-state imaging in biological context in the electron microscope

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

Evaluation of Electron Microscopic Sample Preparation Methods and Imaging Techniques for Characterization of Cell-Mineral Aggregates

Scanning Electron Microscopy (SEM) is used to image geomicrobiological samples, typically containing interfaces between “hard and soft materials” such as minerals and cells, which represent challenges for artifact-free preparation for high-resolution imaging. We used cell-mineral aggregates produced during microbial Fe(II) oxidation and Fe(III) reduction to evaluate different sample preparation and imaging techniques. Both rapid freezing and standard critical point drying (CPD) preserve structures of geomicrobiological samples, at least the ones obtained for Fe(II)-oxidizing and Fe(III)-reducing bacteria, without artifacts. We recommend a SEM sample preparation scheme for geomicrobiological specimens and discuss critical parameters like fixation, dehydration, coating, and acceleration voltages.     

Parakeet: a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography

In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘digital twin’ software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.     

Atomic‐Resolution Imaging of Halide Perovskites Using Electron Microscopy

Atomic‐resolution imaging of halide perovskites (HPs) using transmission electron microscopy (TEM) is challenging because of the sensitivity of their structures to the electron beam. In this article, recent achievements in this area are reviewed, covering both all‐inorganic and organic–inorganic hybrid HPs, with an emphasis on the specific imaging conditions that have proven to be effective in avoiding electron beam‐induced structural damage. The discussion focusses on the total electron dose that HPs can bear before being damaged and the effects of different imaging modes, accelerating voltages, and temperatures. The crucial role of a direct‐detection electron‐counting camera in reducing the required electron dose is outlined, which is indispensable for imaging extremely sensitive organic–inorganic hybrid perovskites. In addition to reviewing published works, the results of initial attempts to perform atomic‐resolution elemental mapping for an all‐inorganic HP and image a hybrid HP using scanning TEM are introduced. The preparation of a TEM specimen from macroscopic crystals or devices of HPs, which is very important for practical applications but has not yet received attention, is also discussed. This article aims to provide guidance on the acquisition of atomic‐resolution TEM images of HPs and inspire the development of more imaging technologies for sensitive materials.     

Single organelle dynamics linked to 3D structure by correlative live‐cell imaging and 3D electron microscopy

Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB‐SEM, which significantly reduces time required for image acquisition and data processing.      

Structural characterisation of islet amyloid polypeptide fibrils

Islet amyloid is found in many patients suffering from type 2 diabetes. Amyloid fibrils found deposited in the pancreatic islets are composed of a 37-residue peptide, known as islet amyloid polypeptide (IAPP) (also known as amylin) and are similar to those found in other amyloid diseases. Synthetic IAPP peptide readily forms amyloid fibrils in vitro and this has allowed fibril formation kinetics and the overall morphology of IAPP amyloid to be studied. Here, we use X-ray fibre diffraction, electron microscopy and cryo-electron microscopy to examine the molecular structure of IAPP amyloid fibrils. X-ray diffraction from aligned synthetic amyloid fibrils gave a highly oriented diffraction pattern with layer-lines spaced 4.7 A apart. Electron diffraction also revealed the characteristic 4.7 A meridional signal and the position of the reflection could be compared directly to the image of the diffracting unit. Cryo-electron microscopy revealed the strong signal at 4.7 A that has been previously visualised from a single Abeta fibre. Together, these data build up a picture of how the IAPP fibril is held together by hydrogen bonded beta-sheet structure and contribute to the understanding of the generic structure of amyloid fibrils.     

Insights into the architecture of the Ure2p yeast protein assemblies from helical twisted fibrils

The protein Ure2 from baker's yeast is associated with a heritable and transmissible phenotypic change in the yeast Saccharomyces cerevisiae. Such prion properties are thought to arise from the fact that Ure2p is able to self-assemble into insoluble fibrils. Assemblies of Ure2p are composed of full-length proteins in which the structure of the globular, functional, C-terminal domain is retained. We have carried out structural studies on full-length, wild-type Ure2p fibrils with a regularly twisted morphology. Using electron microscopy and cryo-electron microscopy with image analysis we show high-resolution images of the twisted filaments revealing details within the fibrillar structure. We examine these details in light of recent proposed models and discuss how this new information contributes to an understanding of the architecture of Ure2p yeast prion fibrils.     

Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage

Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio. Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.     

Correlative 3D imaging of whole mammalian cells with light and electron microscopy

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10–20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.     

Ultrastructure of plant chromosomes by high-resolution scanning electron microscopy

A technique is described for using standard squash preparations of mitotic and meiotic chromosomes for both light microscopy and subsequent high-resolution scanning electron microscopy for investigation of the same specimen. Depending on the microscope and conditions of preparation, a resolution of a few nanometers is routinely possible. Tilting of the specimen provides a three-dimensional insight into chromosomal structures. Combination of material-dependent signals of backscattered electrons with the secondary electron image allows an unambiguous localization of surface markers.     

Automated 100-position specimen loader and image acquisition system for transmission electron microscopy

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the "Gatling", permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 h. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science, and nanotechnology that require rapid screening and image analysis of multiple specimens.     

Structural analysis of vimentin and keratin intermediate filaments by cryo-electron tomography

Intermediate filaments are a large and structurally diverse group of cellular filaments that are classified into five different groups. They are referred to as intermediate filaments (IFs) because they are intermediate in diameter between the two other cytoskeletal filament systems that is filamentous actin and microtubules. The basic building block of IFs is a predominantly alpha-helical rod with variable length globular N- and C-terminal domains. On the ultra-structural level there are two major differences between IFs and microtubules or actin filaments: IFs are non-polar, and they do not exhibit large globular domains. IF molecules associate via a coiled-coil interaction into dimers and higher oligomers. Structural investigations into the molecular building plan of IFs have been performed with a variety of biophysical and imaging methods such as negative staining and metal-shadowing electron microscopy (EM), mass determination by scanning transmission EM, X-ray crystallography on fragments of the IF stalk and low-angle X-ray scattering. The actual packing of IF dimers into a long filament varies between the different families. Typically the dimers form so called protofibrils that further assemble into a filament. Here we introduce new cryo-imaging methods for structural investigations of IFs in vitro and in vivo, i.e., cryo-electron microscopy and cryo-electron tomography, as well as associated techniques such as the preparation and handling of vitrified sections of cellular specimens.     

Correlative single‐molecule localization microscopy and electron tomography reveals endosome nanoscale domains

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction‐limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single‐molecule photo‐switching are opposed. Here, we developed a novel superCLEM workflow that combines triple‐color SMLM (dSTORM & PALM) and electron tomography using semi‐thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub‐compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.