Electron microscopy of the early Caenorhabditis elegans embryo

The early Caenorhabditis elegans embryo is currently a popular model system to study centrosome assembly, kinetochore organization, spindle formation, and cellular polarization. Here, we present and review methods for routine electron microscopy and 3D analysis of the early C. elegans embryo. The first method uses laser‐induced chemical fixation to preserve the fine structure of isolated embryos. This approach takes advantage of time‐resolved fixation to arrest development at specific stages. The second method uses high‐pressure freezing of whole worms followed by freeze‐substitution (HPF‐FS) for ultrastructural analysis. This technique allows staging of developing early embryos within the worm uterus, and has the advantage of superior sample preservation required for high‐resolution 3D reconstruction. The third method uses a correlative approach to stage isolated, single embryos by light microscopy followed by HPF‐FS and electron tomography. This procedure combines the advantages of time‐resolved fixation and superior ultrastructural preservation by high‐pressure freezing and allows a higher throughput electron microscopic analysis. The advantages and disadvantages of these methods for different applications are discussed.     

Aclar discs: a versatile substrate for routine high-pressure freezing of mammalian cell monolayers

High‐pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze‐substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high‐pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high‐pressure freeze, freeze‐substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high‐pressure freezing, freeze‐substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.     

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze‐substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high‐pressure frozen and aldehyde‐fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.     

Contribution of cryofixation and freeze-substitution to analytical microscopy: a study of Tritrichomonas foetus hydrogenosomes

The hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. It is found in several trichomonad species, protists living in oxygen-poor environments, as well as certain free-living ciliates, rumen ciliates, and some fungi. We performed a comparative microanalytical study (energy dispersive X-ray analysis and electron spectroscopic imaging) of different fixation methods for electron microscopy analyzing hydrogenosomes of the bovine parasite Tritrichomonas foetus. The study included the elemental composition and the mapping of calcium, phosphorus, and oxygen. A preparation of T. foetus cells, based on cryoimmobilization by high-pressure freezing and freeze-substitution, was compared to a second preparation based on chemical fixation followed by dehydration and routine processing. The ultrastructural preservation achieved by the cryotechnique was far superior to the chemical fixation, since it allowed the successful cryoimmobilization of intracellular ion contents. The detection of several cations (Al, Mg, Co, Ca, Fe) by X-ray microanalysis inside the peripheral vesicle of the hydrogenosome was only possible in cryofixed cells. The presence of aluminum and cobalt ion in the hydrogenosomal vesicle was established for the first time. Electron-spectroscopic images of calcium showed that this element, in addition to the vesicle compartment, is present in the hydrogenosome's membrane in varying concentrations, which might reflect changes in the physiology of this organelle.     

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at ?150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.     

Morphology visualization of irregular shape bacteria by electron holography and tomography

In the current work, irregular morphology of Staphylococcus aureus bacteria has been visualized by phase retrieval employing off‐axis electron holography (EH) and 3D reconstruction electron tomography using high‐angle annular dark field scanning transmission electron microscopy (HAADF‐STEM). Bacteria interacting with gold nanoparticles (AuNP) acquired a shrunken or irregular shape due to air dehydration processing. STEM imaging shows the attachment of AuNP on the surface of cells and suggests an irregular 3D morphology of the specimen. The phase reconstruction demonstrates that off‐axis electron holography can reveal with a single hologram the morphology of the specimen and the distribution of the functionalized AuNPs. In addition, EH reduces significantly the acquisition time and the cumulative radiation damage (in three orders of magnitude) over biological samples in comparison with multiple tilted electron expositions intrinsic to electron tomography, as well as the processing time and the reconstruction artifacts that may arise during tomogram reconstruction.     

Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C

High‐pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high‐pressure frozen, freeze‐substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.     

LIP-model-based three-dimensional reconstruction and visualization of HIV-infected entire cells

This paper presents a global solution from acquisition to visualization for the three-dimensional reconstruction of cell sections. Original techniques are proposed for the correct handling of the geometrical section distortions, and a new interpretation based on the logarithmic image processing (LIP) model is applied in order to create normalized grey-level sections where these are missing. Finally, a new method for generating a mesh of triangles to describe the envelope of the reconstructed cell is proposed, as well as a visualization mixing image synthesis and grey-level information. The product allows the user to explore the reconstructed cellular block in any desired direction, by showing user-defined grey-level sections inside the block mixed to a synthetic view of the cell envelope.     

Heptane and isooctane as embedding fluids for high-pressure freezing of Petunia ovules followed by freeze-substitution

In comparison with other fixation methods, high-pressure freezing and freeze-substitution of Petunia ovules lead to improved ultrastructural preservation of all tissues. Crucial for adequate high-pressure freezing is the absence of air in the specimen sandwich; air has to be replaced by an embedding fluid. Frequently, 1-hexadecene is used for this purpose. Using 1-hexadecene as an embedding fluid resulted in only 5–10% of Petunia ovules being preserved without disturbance of the ultrastructure due to ice-crystal damage. Since 1-hexadecene is not soluble in acetone at − 90 °C, freeze-substitution is hindered when ovules remained completely surrounded by it; this results in recrystallization when the temperature is raised. We tested and compared the suitability of heptane and isooctane as embedding fluids for high-pressure freezing and freeze-substitution, reasoning that because of their low melting points and low relative densities, phase separation during freeze-substitution would result in complete exposure of the ovules to the substitution medium, leading to adequate freeze-substitution. Using either heptane or isooctane as an embedding fluid yielded up to 90% ice-crystal-free ovules. Both compounds, however, have some damaging effects on the outer one or two cell layers of the ovule, but not on the inner tissues.     

Ultrastructure of the frog retina after high-pressure freezing and freeze substitution

In many types of tissue, high-pressure freezing (HPF), followed by freeze substitution, can produce excellent ultrastructural preservation at depths over 10 times that obtained by other cryofixation techniques. However, in the case of neural tissue, the benefits of HPF have not been realized. In the present study, isolated frog ( Rana pipiens) retina was sliced at a thickness of 150 or 350 μm, rapidly frozen in a Balzers HPM 010 high-pressure freezer, and freeze substituted with 1% OsO₄ and 0.1% tannic acid in acetone. Specially designed HPF chambers and specific freezing media (35% high-MW dextran for 150-μm slices or 15% low-MW dextran for 350-μm slices) were required for adequate freezing.
The quality of preservation after HPF was excellent throughout the retina in both the 150- and 350-μm slices, compared with chemically fixed slices. Specifically, HPF resulted in better preserved cellular, mitochondrial and nuclear membranes in all retinal layers.
This is the first study to successfully cryofix all of the layers of the retina. The increased depths of adequate freezing achieved by HPF should facilitate various ultrastructural studies of retina, as well as of other CNS tissues, where preservation approaching that of the 'native' state is required.     

用数字全息层析成像技术测量毛细管的内径及壁厚

研究了用数字全息层析成像技术测量微毛细管结构的可行性.考虑毛细管具有理想的柱对称结构,因此采用单幅全息图获取到的物光波复振幅数据来模拟不同角度下的投影数据.分别运用滤波反投影重建算法和傅里叶衍射重建算法对微毛细管进行折射率三维重构;根据重构的折射率切片图,进一步运用相关的边缘提取算法处理得到毛细管的内径及壁厚尺寸.实验结果表明,在合理的光路环境设置下,满足Rytov近似条件下的傅里叶衍射重建算法比滤波反投影重建算法更能够正确反映物体的结构尺寸,更适合用于微小弱散射物体的几何参数测量.实验结果验证了用数字全息方法实现衍射层析重建的可行性,从而为具有柱对称结构的弱散射物体的无损测量提供了一种新的途径.     

Surface and moisture characteristics of jute using a D.C. glow discharge argon plasma

A D.C. glow discharge plasma source was employed to characterize changes in water absorbance and surface morphology of natural jute fibers. The low temperature plasma removed moisture from the fibers and significantly modified surface properties. Scanning electron microscopy and energy dispersive X-ray spectroscopy were used for characterization. Open source software was used for the processing of the scanning electron micrographs. The changes in the macromolecular structure and the crystallinity were characterized by X-ray diffraction. Thermogravimetric analysis demonstrated moisture removal from the fibers following plasma treatment.     

Precise observation of C. elegans dynamic behaviours under controlled thermal stimulus using a mobile phone–based microscope

We investigated the temperature‐dependent locomotion of Caenorhabditis elegans by using the mobile phone–based microscope. We developed the customized imaging system with mini incubator and smartphone to effectively control the thermal stimulation for precisely observing the temperature‐dependent locomotory behaviours of C. elegans. Using the mobile phone–based microscope, we successfully followed the long‐term progress of specimens of C. elegans in real time as they hatched and explored their temperature‐dependent locomotory behaviour. We are convinced that the mobile phone–based microscope is a useful device for real time and long‐term observations of biological samples during incubation, and can make it possible to carry out live observations via wireless communications regardless of location. In addition, this microscope has the potential for widespread use owing to its low cost and compact design.     

Cryo-electron microscopy and three-dimensional reconstruction of actin filaments

Actin filaments have been examined by electron microscopy whilst in a frozen-hydrated state. Filaments embedded in a vitreous water layer are basically similar to those prepared by negative staining and show characteristic helical substructure, where the pitches of the helices are about 70 nm and 6 nm. Variability in spacing between long pitch helix cross-over points has been observed, which is consistent with intrinsic angular disorder between successive filament subunits. Fourier transforms of the most ordered filaments show four strong layer lines that index as the first, fifth, sixth and seventh orders of a 35 nm repeat. A three-dimensional helical reconstruction, calculated to a resolution of about 4 nm, shows the individual subunits to be orientated with their long axes roughly perpendicular to the filament axis. Each subunit is somewhat curved and is resolved into two domains. Most connections between successive subunits appear to be made close to the filament axis. We also report on the performance of the specimen holder (Philips PW 5699) used in this work.     

Serial sectioning and digitization of porous media for two- and three-dimensional analysis and reconstruction

A procedure is proposed for serial sectioning of impregnated porous media, for photographing the sections under the microscope, and for digitizing the image using an IBM PC (XT) with a digitizing board. The result is a three-dimensional matrix containing the enhanced digitized image in a binary form of O's and I's which represent solid and pore spaces respectively. The procedure was tested on three different porous materials: (1) a pack of glass beads, 170–350 μm; (2) Berca sandstone; and (3) Elora clay loam soil. Each of these was impregnated, forty-six to eighty cross-sections were prepared by close parallel polishing of 10–50 μm per section, and then the sections were photographed and digitized. Examples of the real and digitized cross-sectioned samples are given as well as an example of reconstructed images in a plane perpendicular to the plane of sampling. It is proposed that the method of serial sectioning and automated digitization of porous media provides a powerful tool for further three-dimensional geometrical and topological investigation of pore space to be used in models of fluid transport phenomena.     

Correlative light and electron microscopy for the analysis of cell division

Correlative light and electron microscopy (CLEM) has recently gained increasing attention, because it enables the acquisition of dynamic as well as ultrastructural information about subcellular processes. It is the power of combining the two imaging modalities that gives additional information as compared to using the imaging techniques separately. Here, we briefly summarize two CLEM approaches for the analysis of cells in mitosis and cytokinesis.     

The in situ architecture of Escherichia coli ribosomal RNA derived by electron spectroscopic imaging and three-dimensional reconstruction

The structures of the large and small ribosomal subunits of Escherichia coli were reconstructed using spectroscopic electron microscopy and quaternion-assisted angular reconstitution to resolutions of better than 4 nm. In addition, the distributions of phosphorus within these complexes were reconstructed. The three-dimensional reconstruction of the distribution of this atomic element is an extension of microanalysis (in two dimensions) for phosphorus identification and mapping, as a signature of the arrangement of the phosphate backbones of the constituent ribosomal RNAs. The results on both the phosphorus reconstructions and the total reconstructions (protein and ribosomal RNA) reveal several passageways through both subunits. The structures correspond favourably with other independent reconstructions of the whole E. coli ribosome from cryoelectron micrographs and their accompanying models of translation (Frank et al ., Nature , 376, 441–444, 1995; Stark et al ., Structure , 3, 815–821, 1995). The overall reconstructions in conjunction with the phosphorus (rRNA) distributions are the first to be achieved synchronously for this nucleoprotein complex.     

Computer modeling of three-dimensional reconstruction algorithm of cathodoluminescence material properties,analysis of errors,and optimization of variable parameters

Three-dimensional (3-D) reconstruction of cathodoluminescence (CL) properties of materials (Saparin et al. 1997) is a new nondestructive technique that measures quantum yield information of subsurface layers as a set of two-dimensional image sections. This technique is able to synthesize a 3-D image without destroying the sample. The basis for this technique is the fact that the electrons in scanning electron microscopy penetrate the sample at different depths with a variation of accelerating voltage. The detected CL emission integrates the information from the full volume occupied by the charge carriers. Estimating the errors that occur during the reconstruction process is complex since these errors are caused by necessary approximations, noise, and the imperfection of the 3-D reconstruction algorithm that is employed. An analysis of the factors that affect the accuracy of the technique has been made. The estimation of systematic errors and the optimization of variable parameters were calculated by computer modeling.     

Study of the structure of the air and blood capillaries of the gas exchange tissue of the avian lung by serial section three-dimensional reconstruction

We have previously reconstructed the gas exchange tissue of the adult muscovy duck, Cairina moschata using a method of manually aligning sections and tracing the contours of the components of the gas exchange tissue. This reconstruction method demonstrated that the air capillaries are comprised of an expanded globular part interconnected by narrow air channels. The blood capillaries completely surround the air capillaries forming an anastomosing meshwork of short segments. However, the resulting reconstruction was limited in scope because of the laborious process of tracing the profiles of each component through the sequence of micrographs. We have now reconstructed a larger proportion of the exchange tissue by using a cross-correlation based alignment strategy and have demonstrated that the staining intensity of each of the exchange tissue components is sufficiently different to allow them to be identified by simple filtering and thresholding. The resulting reconstructions sample a much larger proportion of the exchange tissue and demonstrate the heterogeneity of structures from different locations in the parabronchus. We have shown that a sheet-flow-type arrangement of blood capillaries surrounds the infundibulum; this represents an unexpected functional convergence with the arrangement of blood capillaries surrounding the mammalian alveoli. It is feasible, using this reconstruction strategy, to analyse the exchange tissue of a large number of avian species in order to determine structural correlates of function. The resulting reconstructions could be analysed in order to determine the basis of the functional efficiency and rigidity of the avian lung.     

Comparison of single‐particle analysis and electron tomography approaches: an overview

Three‐dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X‐ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two three‐dimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single‐particle analysis. The first one is currently used to elucidate the three‐dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.