生物三维电子显微学进入全新高速发展时期

生物三维电子显微学主要由三个部分组成——电子晶体学、单颗粒技术和电子断层成像术,其结构解析对象的尺度范围介于x射线晶体学与光学显微镜之间,适合从蛋白质分子结构到细胞和组织结构的解析。以冷冻电镜技术与三维重构技术为基础的低温电子显微学代表了生物电子显微学的前沿。低温单颗粒技术对于高度对称的病毒颗粒的解析最近已达到3．8A分辨率,正在成为解析分子量很大的蛋白质复合体高分辨结构的有效技术手段。低温电子断层成像技术目前对于真核细胞样品的结构解析已达到约40A的分辨率,在今后5年有望达到20A。这样,把x射线晶体学、NMR以及电镜三维重构获得的蛋白质分子及复合体的高分辨率的结构,锚定到较低分辨率的电子断层成像图像中,从而在细胞水平上获得高精确的蛋白质空间定位和原子分辨率的蛋白质相互作用的结构信息。这将成为把分子水平的结构研究与细胞水平的生命活动衔接起来的可行途径。     

Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.     

Processing scarce biological samples for light and transmission electron microscopy

Light microscopy (LM) and transmission electron microscopy (TEM) aim at understanding the relationship structure-function. With advances in biology, isolation and purification of scarce populations of cells or subcellular structures may not lead to enough biological material, for processing for LM and TEM. A protocol for preparation of scarce biological samples is presented. It is based on pre-embedding the biological samples, suspensions or pellets, in bovine serum albumin (BSA) and bis-acrylamide (BA), cross-linked and polymerized. This preparation provides a simple and reproducible technique to process biological materials, present in limited quantities that can not be amplified, for light and transmission electron microscopy.     

A public software for energy filtering transmission electron tomography (EFTET-J): application to the study of granular inclusions in bacteria from Riftia pachyptila

Energy-filtering transmission electron microscopy (EFTEM) allows the determination of elemental distributions out of a sequence of energy filtered images. Combined with electron tomography, EFTEM is a powerful tool to obtain three-dimensional chemical maps from sub-cellular structures. However, there is no existing software in the public-domain for the computation and analysis of 3D-chemical maps. Here, we present a Java-based program to compute 3D-elemental distribution. This program is available as a set of plug-ins for the public-domain Java image processing program Image J inspired by NIH Image. Its implemented algorithms have been successfully applied to the three-dimensional localization of iron granules in semi thin (200 nm) epon sections from the vent worm Riftia pachyptalia.     

Application of electron tomography to fungal ultrastructure studies

Access to structural information at the nanoscale enables fundamental insights into many complex biological systems. The development of the transmission electron microscope (TEM) has vastly increased our understanding of multiple biological systems. However, when attempting to visualize and understand the organizational and functional complexities that are typical of cells and tissues, the standard 2-D analyses that TEM affords often fall short. In recent years, high-resolution electron tomography methods, coupled with advances in specimen preparation and instrumentation and computational speed, have resulted in a revolution in the biological sciences. Electron tomography is analogous to medical computerized axial tomography (CAT-scan imaging) except at a far finer scale. It utilizes the TEM to assemble multiple projections of an object which are then combined for 3-D analyses. For biological specimens, tomography enables the highest 3-D resolution (5 nm spatial resolution) of internal structures in relatively thick slices of material (0.2-0.4 microm) without requiring the collection and alignment of large numbers of thin serial sections. Thus accurate and revealing 3-D reconstructions of complex cytoplasmic entities and architecture can be obtained. Electron tomography is now being applied to a variety of biological questions with great success. This review gives a brief introduction into cryopreservation and electron tomography relative to aspects of cytoplasmic organization in the hyphal tip of Aspergillus nidulans.     

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     

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.     

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.     

High-Contrast Observation of Unstained Proteins and Viruses by Scanning Electron Microscopy

Scanning electron microscopy (SEM) is an important tool for the nanometre-scale analysis of the various samples. Imaging of biological specimens can be difficult for two reasons: (1) Samples must often be left unstained to observe detail of the biological structures; however, lack of staining significantly decreases image contrast. (2) Samples are prone to serious radiation damage from electron beam. Herein we report a novel method for sample preparation involving placement on a new metal-coated insulator film. This method enables obtaining high-contrast images from unstained proteins and viruses by scanning electron microscopy with minimal electron radiation damage. These images are similar to those obtained by transmission electron microscopy. In addition, the method can be easily used to observe specimens of proteins, viruses and other organic samples by using SEM.     

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.     

XMIPP: a new generation of an open-source image processing package for electron microscopy

X-windows based microscopy image processing package (Xmipp) is a specialized suit of image processing programs, primarily aimed at obtaining the 3D reconstruction of biological specimens from large sets of projection images acquired by transmission electron microscopy. This public-domain software package was introduced to the electron microscopy field eight years ago, and since then it has changed drastically. New methodologies for the analysis of single-particle projection images have been added to classification, contrast transfer function correction, angular assignment, 3D reconstruction, reconstruction of crystals, etc. In addition, the package has been extended with functionalities for 2D crystal and electron tomography data. Furthermore, its current implementation in C++, with a highly modular design of well-documented data structures and functions, offers a convenient environment for the development of novel algorithms. In this paper, we present a general overview of a new generation of Xmipp that has been re-engineered to maximize flexibility and modularity, potentially facilitating its integration in future standardization efforts in the field. Moreover, by focusing on those developments that distinguish Xmipp from other packages available, we illustrate its added value to the electron microscopy community.     

Bovine olfactory and nasal respiratory epithelium surfaces

Summary High-voltage transmission electron microscopy and cryo-ultramicrotomy together with scanning electron microscopy and some conventional transmission electron microscopy of ultrathin sections have been applied to the mucous surfaces of bovine olfactory and respiratory epithelia. Distal segments of olfactory cilia tend to run in parallel and could be followed over distances up to about 30 m using high-voltage electron microscopy. This technique and scanning electron microscopy showed that on average 12–13 of such cilia could be observed per nerve ending. After correction for obscured cilia this number becomes about 17. High-voltage micrographs and micrographs made from sections prepared with a cryo-ultramicrotome showed the presence of electron-lucent pockets inside the olfactory mucus. The latter technique also showed that the mucus itself is not fibrous, but rather a continuum varying in electron density. The mucus layer contains various granular structures. Ciliary and microvillar membranes appear thicker with cryo-ultramicrotomy than when the sections are prepared with conventional techniques. The cores of the axonemal microtubules in olfactory as well as in respiratory cilia are darkly stained with this technique. Vesicles present inside the nerve endings are also darkly stained. Dimensions and some other numerical values of interest in olfaction are presented.     

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。     

Low-Resolution Density Maps from Atomic Models: How Stepping “Back” Can Be a Step “Forward”

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15–40 Å), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.     

An electron microscopic study of the ultrastructure of microbial cells in extreme biotopes in situ

The ultrastructure of microbial cells was studied in situ in natural biotopes by high-resolution transmission electron microscopy using the known methods of cryofractography, thin sectioning, and the negative staining of total cell specimens, as well as the new methods of the low-temperature fractionation of microbial cells (providing for the recovery of cells from natural sources and their concentration), the preparation of micromonoliths, and aimed electron microscopy. Among the natural biotopes studied were permafrost ground and oil sludge. Most of the microorganisms found in the 1- to 3-million-year-old permafrost ground were represented by resting forms (spores, cysts, and cystlike cells with specific organomineral envelopes). Oil sludge older than 35 years contained bacteria of atypical morphology and ultrastructure, including various resting forms and ultramicrobacteria. The data obtained is indicative of considerable promise of high-resolution electron microscopy for studying microbial communities in situ.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 832–840.Original Russian Text Copyright © 2004 by Dmitriev, Suzina, Barinova, Duda, Boronin.     

The three-dimensional structure of the cyanobacterium Synechocystis sp. PCC 6803

To advance our knowledge of the model cyanobacterium Synechocystis sp. PCC 6803 we investigated the three-dimensional organization of the cytoplasm using standard transmission electron microscopy and electron tomography. Electron tomography allows a resolution of ~5 nm in all three dimensions, superior to the resolution of most traditional electron microscopy, which is often limited in part by the thickness of the section (70 nm). The thylakoid membrane pairs formed layered sheets that followed the periphery of the cell and converged at various sites near the cytoplasmic membrane. At some of these sites, the margins of thylakoid membranes associated closely along the external surface of rod-like structures termed thylakoid centers, which sometimes traversed nearly the entire periphery of the cell. The thylakoid membranes surrounded the central cytoplasm that contained inclusions such as ribosomes and carboxysomes. Lipid bodies were dispersed throughout the peripheral cytoplasm and often juxtaposed with cytoplasmic and thylakoid membranes suggesting involvement in thylakoid maintenance or biogenesis. Ribosomes were numerous and mainly located throughout the central cytoplasm with some associated with thylakoid and cytoplasmic membranes. Some ribosomes were attached along internal unit-membrane-like sheets located in the central cytoplasm and appeared to be continuous with existing thylakoid membranes. These results present a detailed analysis of the structure of Synechocystis sp. PCC 6803 using high-resolution bioimaging techniques and will allow future evaluation and comparison with gene-deletion mutants.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Zernike phase contrast cryo-electron tomography of whole mounted frozen cells

Cryo-electron tomography of frozen hydrated cells has provided cell biologists with an indispensable tool for delineating three-dimensional arrangements of cellular ultrastructure. To avoid the damage induced by electron irradiation, images of frozen hydrated biological specimens are generally acquired under low-dose conditions, resulting in weakly contrasted images that are difficult to interpret, and in which ultrastructural details remain ambiguous. Zernike phase contrast transmission electron microscopy can improve contrast, and can also fix a fatal problem related to the inherent low contrast of conventional electron microscopy, namely, image modulation due to the unavoidable setting of deep defocus. In this study, we applied cryo-electron tomography enhanced with a Zernike phase plate, which avoids image modulation by allowing in-focus setting. The Zernike phase contrast cryo-electron tomography has a potential to suppress grainy background generation. Due to the smoother background in comparison with defocus phase contrast cryo-electron tomography, Zernike phase contrast cryo-electron tomography could yield higher visibility for particulate or filamentous ultrastructure inside the cells, and allowed us to clearly recognize membrane protein structures.     

Reprint of “Stereology meets electron tomography: towards quantitative 3D electron microscopy” [J. Struct. Biol. 159 (2007) 443–450]

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists.This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2–5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method.Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.