Techniques and applications of three‐dimensional electron microscopy in entomological research

Structural studies using two‐dimensional (2D) images show limitations in understanding the structure and functions of cellular organelle and protein. To overcome the difficulty, over the last few years 3D reconstruction techniques using electron microscopy have been developed at extremely high speed. In this paper, currently available 3D reconstruction techniques of electron microscopy (such as electron tomography, serial section analysis and single particle analysis) are introduced using our data as examples of the application. The 3D structure of mitochondria with the defect of mitochondrial protein in round worm, Caenorhabditis elegans, through electron tomography, the cell–cell interaction in lamina of Drosophila melanogaster by serial‐section using ultramicrotome and high‐voltage electron microscopy and a thin filament related to muscle contraction in Drosophila melanogaster were used for examples of the application. These results through 3D reconstruction reveal the structural changes in a cellular organelle and protein that had not been shown by 2D structure.     

基于计算机三维图象技术的失语症病灶空间定位法

首次把计算机三维重建方法应用于失语症的研究中,主要有三方面的工作:(1)建立了一套精细程度较高的标准CT脑图,用三维重建方法建立了标准立体头颅,并用伪彩色显示不同的脑结构和语言区.(2)提出标准化应在三维空间内进行的观点,改进的标准化方法充分利用了CT所能提供的关于脑结构的信息,从而使病灶在每层标准CT脑图上的定位更加准确.(3)对两类临床表现不同但CT显示病灶位置相近的失语症病人共47例进行了研究.通过对标准化之后的病灶的统计处理,得到病灶的集中部位,并对其进行三维重建,在标准立体头颅中显示其空间位置的差别.     

Three‐dimensional ultrastructure of chloroplast pockets formed under salinity stress

We investigated the invagination structure of a chloroplast that surrounds organelles such as mitochondria and peroxisomes within a thin layer of chloroplast stroma, which is called a chloroplast pocket. In this study, chloroplast pockets were observed in rice plants subjected to salinity stress but not under moderate growth condition. They included cytosol, transparent structure, lipid bodies, mitochondria, and peroxisomes. We constructed the three‐dimensional architecture of chloroplast pockets by using serial images obtained by transmission electron microscopy and focused ion beam‐scanning electron microscopy. Three types of chloroplast pockets were observed by transmission electron microscopy: Organelles were completely enclosed in a chloroplast pocket (enclosed type), a chloroplast pocket with a small gap in the middle part (gap type), and a chloroplast pocket with one side open (open type). Of the 70 pockets observed by serial imaging, 35 were enclosed type, and 21 and 14 were gap and open types, respectively. Mitochondria and peroxisomes were often in contact with the chloroplast pockets. Focused ion beam‐scanning electron microscopy revealed chloroplasts with a sheet structure partially surrounding peroxisomes. This fact suggests that chloroplasts might construct large sheet structures that would be related to the formation of chloroplast pockets.     

Insect morphology in the age of phylogenomics: innovative techniques and its future role in systematics

A brief account of the history of insect morphology is given. Different techniques and analytical methods used in current projects on insect morphology and phylogeny and their optimized combined application are described. These include fixation, dissection, maceration, histology (microtome sectioning), scanning electron microscopy (SEM), transmission electron microscopy (TEM), serial block‐face scanning electron microscopy (SBFSEM), focused ion beam scanning electron microscopy (FIB/SEM), confocal laser scanning microscopy (CLSM), bleaching, micro‐computed tomography (μCT), computer‐based three‐dimensional reconstruction, focus stacking of digital images, geometric morphometrics and the storage of morphological metadata. The role of insect morphology in the “age of phylogenomics” is discussed.     

Use of confocal laser scanning microscopy on soil thin-sections for improved characterization of microbial growth in unconsolidated soils and aquifer materials

Confocal laser scanning microscopy (CLSM) was utilized to examine samples from an aquifer microcosm that was used to investigate microbially mediated losses in hydraulic conductivity. Samples were fixed, dehydrated and dried to prepare the biological material in a fashion similar to that used previously for viewing under the scanning electron microscope. Then, samples were prepared as thin-sections by employing soil micromorphological techniques. Serial images generated by the CLSM technique were visualized using computer three-dimensional rendering software. Results from the CLSM technique were compared with simple fluorescence microscopy of thin-sections and scanning electron microscopy (SEM) of samples from the microcosm. Computer visualization of serial sections with the CLSM technique provided images on a submicron scale in three dimensions. SEM has a much higher resolution, on a nanometer scale, but the results are not three dimensional. Artifacts associated with thin-section preparation are minimal for natural porous media composed mostly of sand, such as aquifer materials. Also, CLSM images are affected minimally by changes to biological material due to sample preparation, whereas artifacts associated with SEM images are very prominent, due to the higher magnification and resolution. CLSM of thin-sections and SEM are very powerful methods for viewing microbial growth in natural porous media, but CLSM is preferable because it allows three-dimensional visualization and measurements of cells and aggregates with few artifacts.     

Computer-aided stereographic representation of an object reconstructed from micrographs of serial thin sections

A method is described which allows a three-dimensional object to be reconstructed from micrographs of serial thin sections using computer graphic techniques. The reconstructed object, which can be rotated three- dimensionally, is displayed on a colour visual display unit and the surface of the object is shaded in order that it can be observed to provide an illusion of a three-dimensional structure. Moreover, the technique makes it possible to represent an inner structure when seen through an outer one, also to observe other sectioned face views. The method as described here allows rapid visual evaluation of the results of three-dimensional reconstruction from serial thin sections when recorded with the aid of a light or an electron microscope.     

三维电子显微镜方法进展

从生物样品（细胞或大分子）的电镜图象重组其三维结构的方法近年取得了重大进展,这是冷冻电镜样品制备、电镜设备、图象处理和分析方法等几方面进步的综合结果。三维电镜方法的进步和完善,使细胞和学家得以了解在复杂的细胞过程中各种相关细胞器之间的空间关系,而使分子生物学家不仅可以研究那些能够形成二维结晶的样品,并为分析具有重要生物功能但不能形成二维结晶的大分子或分子聚休物的结构提供了一种强大的手段。     

PSI: indexing protein structures for fast similarity search

MOTIVATION: We consider the problem of finding similarities in protein structure databases. Current techniques sequentially compare the given query protein to all of the proteins in the database to find similarities. Therefore, the cost of similarity queries increases linearly as the volume of the protein databases increase. As the sizes of experimentally determined and theoretically estimated protein structure databases grow, there is a need for scalable searching techniques. RESULTS: Our techniques extract feature vectors on triplets of SSEs (Secondary Structure Elements). Later, these feature vectors are indexed using a multidimensional index structure. For a given query protein, this index structure is used to quickly prune away unpromising proteins in the database. The remaining proteins are then aligned using a popular alignment tool such as VAST. We also develop a novel statistical model to estimate the goodness of a match using the SSEs. Experimental results show that our techniques improve the pruning time of VAST 3 to 3.5 times while maintaining similar sensitivity.     

Molecular modeling approaches for determining gene function: application to a putative poly-A binding protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 amino acids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.     

Three-dimensional diffractive imaging for crystalline monolayers with one-dimensional compact support

The use of a compact support constraint along the beam direction is considered as a solution to the phase problem for diffraction by two-dimensional protein crystals. Specifically we apply the iterative Gerchberg-Saxton-Fienup algorithm to simulated three-dimensional transmission electron diffraction data from monolayer organic crystals. We find that oversampling along the reciprocal-lattice rods (relrods) normal to the monolayer alone does not solve the phase problem in this geometry in general. However, based on simulations for a crystalline protein monolayer (lysozyme), we find that convergence is obtained in three dimensions if phases are supplied from a few high resolution electron microscope images recorded at small tilts to the beam direction. In the absence of noise, amplitude-weighted phase residuals of around 5 degrees, and a cross-correlation coefficient of 0.96 between the true and estimated potential are obtained if phases are included from images at tilts of up to 15 degrees. The performance is almost as good in the presence of noise at a level that is comparable to that commonly observed in electron crystallography of proteins. The method should greatly reduce the time and labor needed for data acquisition and analysis in cryo-electron microscopy of organic thin crystals by avoiding the need to record images at high tilt angles.     

Building the atomic model for the bacterial flagellar filament by electron cryomicroscopy and image analysis

The bacterial flagellar filament is a helical propeller for bacterial locomotion. It is a well-ordered helical assembly of a single protein, flagellin, and its tubular structure is formed by 11 protofilaments, each in either of the two distinct conformations, L- and R-type, for supercoiling. We have been studying the three-dimensional structures of the flagellar filaments by electron cryomicroscopy and recently obtained a density map of the R-type filament up to 4 angstroms resolution from an image data set containing only about 41,000 molecular images. The density map showed the features of the alpha-helical backbone and some large side chains, which allowed us to build the complete atomic model as one of the first atomic models of macromolecules obtained solely by electron microscopy image analysis (Yonekura et al., 2003a). We briefly review the structure and the structure analysis, and point out essential techniques that have made this analysis possible.     

Organisation spatiale des chloroplastes chezEuglena à l'aide de coupes sériées semi-fines

Summary Euglena, gracilis chloroplasts and mitochondria can be seen as a network. This diverticulated structure of the plastidome is only observed in non-dividing cells, during the cell cycle or during the stationnary phase of growth. We have used successions of serial sectioning (0.5 m thick) to reconstitute chloroplast network in three dimensional model.     

Structure brings clarity: Structured illumination microscopy in cell biology

Biological samples are three dimensional and, therefore, optical sectioning is mandatory for microscopic images to precisely show the localization or function of structures within biological samples. Today, researchers can choose from a variety of methods to obtain optical sections. This article focuses on structured illumination microscopy, which is a group of techniques utilizing a combination of optics and mathematics to obtain optical sections: A structure is imaged onto the sample by optical means and the additional information thereby encoded in the image is used to calculate an optical section from several acquired images. Different methods of structured illumination microscopy (mainly grid projection and aperture correlation) are discussed from a practical point of view, concentrating on advantages, limitations and future prospects of these techniques and their use in cell biology. Structured illumination can also be used to obtain super-resolution information if structures of higher frequency are projected onto the sample. This promising approach to super-resolution microscopy is also briefly discussed from a user's perspective.     

The meiospore ofAllomyces

Summary The arrangement of cellular organelles within the meiospore ofAllomyces macrogynus was found to be similar to the zoospore of this species, with the exception that the meiospore contains membrane enclosed electron dense reserve material which has the appearance of the gamma bodies observed in the zoospores ofBlastocladiella. The three dimensional structure of the side body complex is analyzed with serial sections and compared to homologous organelles in other members of theChytridiomycetes.     

在三维结构上对百合花粉母细胞actin的免疫定位

传统的切片仅仅能够显示样品的平面结构,不能用于细胞中三维网络结构的研究。笔者在DGD(diethylene glycol distearate)包埋去包埋的基础上,结合电镜免疫胶体金技术对大卫百合花粉母细胞胞间及胞内细胞的骨架系统进行了研究,观察到高反差细胞微梁结构的三维网络,actin这一细胞骨架的主要成员被定位在该微梁结构纤维上。三维结构上的研究表明,actin不但是植物细胞核及细胞质骨架的成员,而且也存在于胞间连接结构(胞质桥和胞间连丝)中,推测它可能与细胞融合有关。实验结果同时表明,三维结构免疫胶体金技术对于细胞骨架和核基质的结构蛋白研究是行之有效的。     

Collagen fibril bundles: a branching assembly unit in tendon morphogenesis

The assembly, deposition and organization of collagen fibril bundles and their composite fibrils were studied during morphogenesis of the chick embryo tendon using electron microscopy, serial sections and computer-assisted three-dimensional reconstruction techniques. The 14-day chick embryo is a stage when tendon architecture is being established and rapid changes in the mechanical properties occur between days 14 and 17 of development. Tendon matrix structure develops from discrete subunits, bundles of collagen fibrils. The bundles branch; undergo a gradual rotation over several micrometers; are intimately associated with the cellular elements of the developing tendon; and form arborizing networks within and among fascicles. The organization of discrete fibril segments into bundles, during the establishment of tendon architecture and function, where the segmental fibrillar components could interact with the interfibrillar matrix as well as with adjacent fibrils would contribute to the stabilization of this structure. The observed gradual rotation of the bundles would serve to stabilize the immature bundle through the physical twining of the composite fibrils while the extensive branching of the bundles observed at 14-days of development and their intimate association with the cellular elements would provide a higher order of structure stabilization.     

Method: automatic segmentation of mitochondria utilizing patch classification,contour pair classification,and automatically seeded level sets

Background  

While progress has been made to develop automatic segmentation techniques for mitochondria, there remains a need for more accurate and robust techniques to delineate mitochondria in serial blockface scanning electron microscopic data. Previously developed texture based methods are limited for solving this problem because texture alone is often not sufficient to identify mitochondria. This paper presents a new three-step method, the Cytoseg process, for automated segmentation of mitochondria contained in 3D electron microscopic volumes generated through serial block face scanning electron microscopic imaging. The method consists of three steps. The first is a random forest patch classification step operating directly on 2D image patches. The second step consists of contour-pair classification. At the final step, we introduce a method to automatically seed a level set operation with output from previous steps.     

Ultrastructure of the eukaryotic aminoacyl-tRNA synthetase complex derived from two dimensional averaging and classification of negatively stained electron microscopic images

Several aminoacyl-tRNA synthetases in higher eukaryotes are consistently isolated as a multi-enzyme complex for which little structural information is yet known. This study uses computational methods for analysis of electron microscopic images of the particle. A data set of almost 2000 negatively stained images was processed through reference-free alignment and multivariate statistical analysis. Interpretable structural information was evident in five eigenvectors. Hierarchical ascendant classification extracted clusters corresponding to distinct image orientations. The class averages are consistent with rotations around and orthogonal to a central particle axis and provide particle measurements: approximately 25 nm in height, 30 nm at the widest point and 23 nm thick. The results also provide objective evidence in support of the working structural model and demonstrate the feasibility of obtaining the three dimensional structure of the multisynthetase complex by single particle reconstruction methods.     

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。