Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S(3)EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 μm(3) volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S(3)EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S(3)EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.     

Aerodynamics of saccate pollen and its implications for wind pollination

Pollen grains of many wind-pollinated plants contain 1-3 air-filled bladders, or sacci. Sacci are thought to help orient the pollen grain in the pollination droplet. Sacci also increase surface area of the pollen grain, yet add minimal mass, thereby increasing dispersal distance; however, this aerodynamic hypothesis has not been tested in a published study. Using scanning electron and transmission electron microscopy, mathematical modeling, and the saccate pollen of three extant conifers with structurally different pollen grains (Pinus, Falcatifolium, Dacrydium), we developed a computational model to investigate pollen flight. The model calculates terminal settling velocity based on structural characters of the pollen grain, including lengths, widths, and depths of the main body and sacci; angle of saccus rotation; and thicknesses of the saccus wall, endoreticulations, intine, and exine. The settling speeds predicted by the model were empirically validated by stroboscopic photography. This study is the first to quantitatively demonstrate the adaptive significance of sacci for the aerodynamics of wind pollination. Modeling pollen both with and without sacci indicated that sacci can reduce pollen settling speeds, thereby increasing dispersal distance, with the exception of pollen grains having robust endoreticulations and those with thick saccus walls. Furthermore, because the mathematical model is based on structural characters and error propagation methods show that the model yields valid results when sample sizes are small, the flight dynamics of fossil pollen can be investigated. Several fossils were studied, including bisaccate (Pinus, Pteruchus, Caytonanthus), monosaccate (Gothania), and nonsaccate (Monoletes) pollen types.     

3D gold in situ labelling in the EM

We have developed a novel pre-embedding in situ hybridization labelling method for electron microscopy which has given much greater sensitivity and higher labelling levels than have been achieved previously, together with good ultrastructural preservation. Vibratome sections of plant tissue were labelled throughout their thickness with 1 nm gold antibodies and then silver enhanced, embedded in resin and sectioned for electron microscopy. Because the labelling extends throughout the depth of the specimen, this method permits the study of the 3D arrangement of the labelling at the electron microscope level by either stereo-pair recording, tomographic reconstruction or 3D reconstruction from serial sections. In this paper we describe the application of this method to study the organization of rDNA in pea root tissue.     

云南松花粉形态研究

在云南松(Pinus yunnanensis Fr.)小孢子发生发育过程中,花粉母细胞、四分孢子及花粉粒均见有粘连现象。花粉气囊的形态、大小变化复杂多样。除一般具两个正常气囊的花粉粒外,还观察到气囊不发育、具一个气囊、二个异形气囊、三个气囊和四个气囊的花粉粒。成熟花粉壁从外至内可分为外壁外层、外壁内层、内壁外层和内壁内层,它们的构成成分及形态均有明显差别。贮存后花粉的内壁结构发生了明显变化。     

Microsporogenesis and microgametogenesis of the Argentinian species of Podocarpus (Podocarpaceae)

The development of the microsporangium and male gametophyte of three species of Podocarpus was studied with light microscopy (LM) and the morphology of pollen with scanning and transmission electron microscopy (SEM and TEM). During early stages, the male cone is covered with coriaceous scales. The archesporid cells go through a dormant period. Later the pollen mother cells differentiate and undergo meiosis. Callose is detected around the tetrad and between each monad. The microspore nucleus divides several times to give rise to a multicellular gametophyte, which includes the tube cell, the stalk and body cells, and four prothallial cells. The exine of the pollen grain is rugulate in the corpus and quite smooth in the sacci. The ultrastructure of the pollen wall consists of the alveolate sexine, the laminate nexine I and the amorphous nexine II. The intine is very thin. Comparison of the mature grain of some fossils with living members of the Podocarpaceae reveals great similarity.     

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.     

Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB–SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce “keyframe imaging” as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10⁹ in a single automated experimental run.     

Rapid Three-Dimensional Reconstruction at the Light Microscopic Level and a Technique for Re-Embedding the Same Semithin Sections for Electron Microscopic Examination

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 μm sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.     

On the ultrastructural organization of Trypanosoma cruzi using cryopreparation methods and electron tomography

The structural organization of Trypanosoma cruzi has been intensely investigated by different microscopy techniques. At the electron microscopy level, bi-dimensional analysis of thin sections of chemically fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation. In contrast, more sophisticated and elaborate techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. In addition, the 3D characterization of such cells has been carried out mostly using 3D reconstruction from serial sections, currently considered a low resolution technique when compared to electron tomography (ET). In this work, we re-visited the 3D ultrastructure of T. cruzi using a combination of two approaches: (1) analysis of both conventionally processed and cryofixed and freeze substituted cells and (2) 3D reconstruction of large volumes by serial electron tomography. The analysis of high-pressure frozen and freeze substituted parasites showed novel characteristics in a number of intracellular structures, both in their structure and content. Organelles generally showed a smooth and regular morphology in some cases presenting a characteristic electron dense content. Ribosomes and new microtubule sets showed an unexpected localization in the cell body. The improved preservation and imaging in 3D of T. cruzi cells using cryopreparation techniques has revealed some novel aspects of the ultrastructural organization of this parasite.     

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.     

Invertebrate myosin filament: Parallel subfilament arrangement in the wall of solid filaments from the honeybee, Apis mellifica

Transverse serial sections (100-140 nm thick) of solid myosin filaments of the honeybee, Apis mellifica, were photographed in a JEM-200 electron microscope at 200 kV. The images were digitized and computer processed by rotational filtering. 87% of the myosin filaments showed 6-fold symmetry in their power spectra, confirming the results of earlier works (Beinbrech et al., 1988, 1991). To determine if the subfilaments were arranged parallel to the filament backbone, two methods were used. First, the three images of each myosin filament in the three serial sections were superimposed. 85% of the resulting images showed a strong peak for 6-fold symmetry and the averaged images showed 6 pairs of subfilaments, which gives evidence for parallel arrangement of the subfilaments relative to the filament axis. This result was confirmed by the second method in which a 3-dimensional reconstruction was made. An average image was made from the images of the same 17 myosin filaments from each of the three sections. The data for the 3-dimensional reconstruction were collected by tracing the outlines of the structures in the three successive sections. The resulting stereo image shows a parallel arrangement of the subfilaments.     

Volume scanning electron microscopy for imaging biological ultrastructure

Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre‐scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error‐prone manual tasks of cutting and handling large numbers of sections, and imaging them one‐by‐one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z‐resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block‐face electron microscopy (SBEM), focused ion beam SEM (FIB‐SEM) and automated tape‐collecting ultramicrotome SEM (ATUM‐SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.     

Serial block face scanning electron microscopy—the future of cell ultrastructure imaging

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.     

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.     

茴芹属16种植物的花粉形态及系统学分析

利用光学显微镜和扫描电镜对茴芹属16种植物的花粉形态进行了观察,其中12种为首次报道。结果表明,茴芹属16种植物花粉大小为（19.75～33.03）μm×（11.52～17.41）μm,极轴与赤道轴的比值（P/E）为1.40～2.28,体积大小指数（P×E）^1/2为16.71～23.97,属于中型花粉,具三孔沟,萌发孔位置为边萌发孔。花粉粒赤道面观有近矩形、赤道收缩形、椭圆形和近菱形等类型,其中近矩形和赤道收缩形占多数;极面观为三角形或三角圆形。扫描电镜下,其外壁表面纹饰赤道面观为脑纹状,极面观近光滑或为穴纹状。在伞形科芹亚科中,茴芹属植物的花粉具有中等进化、比较进化的形态特征。研究认为,花粉形态可为个别物种的分类处理提供孢粉学证据。     

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.     

Pollination of Picea orientalis (Pinaceae): saccus morphology governs pollen

Sacci of conifer pollen do not function primarily to increase the efficiency of wind pollination as is widely thought. Rather, they are bladders and cause pollen to float upwards in a liquid drop into the ovules. This observation is seemingly unsupported in the case of oriental spruce (Picea orientalis (L.) Link), which has saccate pollen. Ovulate cones are pendant at the time of pollination, which requires that pollen sink into the ovules. Pollen of oriental spruce floats at first but within 1-2 min sinks into the ovule. As sinking does not occur in saccate pollen of other Pinaceae, a variety of techniques was used to determine anatomical differences leading to this uncharacteristic tendency. Light, scanning electron, and confocal microscopy of the pollen surface yielded no significant appearing difference between pollen of oriental spruce and white spruce. However, transmission electron microscopy of freeze-fixed/freeze-substituted hydrated pollen revealed that the ektexine of oriental spruce pollen sacci is porous compared to that of white spruce. Confocal microscopy allowed examination of pollen hydration dynamics. Water enters pollen at the distal pole between sacci, and resulting rapid expansion of the tube cell forces air out of the saccate space. White spruce pollen remains buoyant because of enclosed air pockets in the saccus ektexine. Evolutionary change in pollen wall anatomy with resultant loss of saccus function is correlated with a change in ovulate strobilus orientation at pollination in oriental spruce. A suite of characters interact in the conifer pollination mechanism, and concerted change in these characters may lead to speciation.     

A Reference-Free Method for Brightness Compensation and Contrast Enhancement of Micrographs of Serial Sections

Three-dimensional (3D) reconstruction of an organ or tissue from a stack of histologic serial sections provides valuable morphological information. The procedure includes section preparation of the organ or tissue, micrographs acquisition, image registration, 3D reconstruction, and visualization. However, the brightness and contrast through the image stack may not be consistent due to imperfections in the staining procedure, which may cause difficulties in micro-structure identification using virtual sections, region segmentation, automatic target tracing, etc. In the present study, a reference-free method, Sequential Histogram Fitting Algorithm (SHFA), is therefore developed for adjusting the severe and irregular variance of brightness and contrast within the image stack. To apply the SHFA, the gray value histograms of individual images are first calculated over the entire image stack and a set of landmark gray values are chosen. Then the histograms are transformed so that there are no abrupt changes in progressing through the stack. Finally, the pixel gray values of the original images are transformed into the desired ones based on the relationship between the original and the transformed histograms. The SHFA is tested on an image stacks from mouse kidney sections stained with toluidine blue, and captured by a slide scanner. As results, the images through the entire stack reveal homogenous brightness and consistent contrast. In addition, subtle color differences in the tissue are well preserved so that the morphological details can be recognized, even in virtual sections. In conclusion, compared with the existing histogram-based methods, the present study provides a practical method suitable for compensating brightness, and improving contrast of images derived from a large number of serial sections of biological organ.     

Applications of a bilateral denoising filter in biological electron microscopy

Due to the sensitivity of biological sample to the radiation damage, the low dose imaging conditions used for electron microscopy result in extremely noisy images. The processes of digitization, image alignment, and 3D reconstruction also introduce additional sources of noise in the final 3D structure. In this paper, we investigate the effectiveness of a bilateral denoising filter in various biological electron microscopy applications. In contrast to the conventional low pass filters, which inevitably smooth out both noise and structural features simultaneously, we found that bilateral filter holds a distinct advantage in being capable of effectively suppressing noise without blurring the high resolution details. In as much, we have applied this technique to individual micrographs, entire 3D reconstructions, segmented proteins, and tomographic reconstructions.     

Automated Reconstruction Algorithm for Identification of 3D Architectures of Cribriform Ductal Carcinoma In Situ

Ductal carcinoma in situ (DCIS) is a pre-invasive carcinoma of the breast that exhibits several distinct morphologies but the link between morphology and patient outcome is not clear. We hypothesize that different mechanisms of growth may still result in similar 2D morphologies, which may look different in 3D. To elucidate the connection between growth and 3D morphology, we reconstruct the 3D architecture of cribriform DCIS from resected patient material. We produce a fully automated algorithm that aligns, segments, and reconstructs 3D architectures from microscopy images of 2D serial sections from human specimens. The alignment algorithm is based on normalized cross correlation, the segmentation algorithm uses histogram equilization, Otsu's thresholding, and morphology techniques to segment the duct and cribra. The reconstruction method combines these images in 3D. We show that two distinct 3D architectures are indeed found in samples whose 2D histological sections are similarly identified as cribriform DCIS. These differences in architecture support the hypothesis that luminal spaces may form due to different mechanisms, either isolated cell death or merging fronds, leading to the different architectures. We find that out of 15 samples, 6 were found to have 'bubble-like' cribra, 6 were found to have 'tube-like' criba and 3 were 'unknown.' We propose that the 3D architectures found, 'bubbles' and 'tubes', account for some of the heterogeneity of the disease and may be prognostic indicators of different patient outcomes.