A Single Method for Cryofixation and Correlative Light, Electron Microscopy and Tomography of Zebrafish Embryos

The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples.     

Staining of Bacteriophages for Light Microscopy

Bacteriophages can be stained with a flagella stain, making them visible by light microscopy.     

The Foundations of High-Frequency Hearing in Early Mammals

It has become common in the paleontological literature to assume that the presence of secondary bony laminae in the cochleae of early mammals indicates that these species were able to perceive high sound frequencies (>20 kHz). This review examines the validity of this idea in the context of comparative physiological data from extant amniotes and surveys a number of unique features of mammalian cochleae that correlate with high-frequency hearing. As it is difficult to imagine how all of these features could have arisen simultaneously, high-frequency hearing probably had a more gradual origin. This suggests that the presence or absence of secondary laminae should be interpreted with greater caution.     

Wrinkle-Free Plastic Sections for Light Microscopy

Plastic sections 0.5 to 2 μm thick are routinely used for light microscopy. Although plastic sections have several advantages over paraffin or celloidin sections, a problem that is often encountered with plastic sections is wrinkling (Fig. 1). Wrinkling occurs during staining when sections dried on glass slides are covered with stain and heated to hasten the penetration of the stain. Mounted sections heated on glass slides, but not stained, ordinarily lack wrinkles, even when examined with phase contrast optics. Similarly, mounted sections covered with stain, but not heated, lack wrinkles; unfortunately, such sections fail to stain adequately. Unmounted sections floated on heated drops of stain also lack wrinkles (Millonig 1980). Thus, it is clear that wrinkling occurs only when mounted sections are covered with stain and heated.     

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins^1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N₂; <2 ppm O₂) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.     

Plastic Sectioning for Light Microscopy of Pyrenomycetes

In preparation for light microscopy, ascocarps of Sordaria fimicola Ces. & DeNot. were embedded in Spurr's medium and sectioned at 1-1.5 μm on an ultramicrotome. Sections were floated on Giemsa staining solution at 60 C for 10-30 min, washed in distilled water, affixed to slides by drying, and mounted in immersion oil. Best preservation of the delicate sterile tissues of the centrum was obtained by fixation in 1% KMnO₄ for 2.5-3 hr, followed by the Giemsa stain. This method is suggested for future studies on the morphology of perithecial ascomycetes.     

A Study of Criteria Permitting the Use of Plastinated Specimens for Light and Electron Microscopy

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultra-structural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.     

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.     

以三维荧光反卷积显微技术研究活体细胞中分泌囊泡的空间分布

获得活体细胞三维图像以观察细胞内分泌囊泡的空间分布有助于细胞分泌机制的研究。三维荧光反卷积显微技术可以为活体细胞观察提供低荧光漂白 ,低毒副作用的快速三维成像。研究了显微成像系统实验测定和理论计算点扩展函数之间的关系 ,并且实验验证了NA 1.6 5物镜条件下 ,理论计算点扩展函数可以较好地反映显微成像系统的特性。然后使用已知物理结构的三维样本对反卷积算法的有效性进行了研究。进而对使用吖啶橙(acridineorange)标记的大鼠胰腺 β细胞分泌囊泡进行观察。结果显示 ,反卷积算法可以有效地去除原始图像中因为焦外光影响产生的模糊 ,处理后图像清晰地显示了细胞内分泌囊泡的空间分布     

Rapid Reconstruction of 3D Neuronal Morphology from Light Microscopy Images with Augmented Rayburst Sampling

Digital reconstruction of three-dimensional (3D) neuronal morphology from light microscopy images provides a powerful technique for analysis of neural circuits. It is time-consuming to manually perform this process. Thus, efficient computer-assisted approaches are preferable. In this paper, we present an innovative method for the tracing and reconstruction of 3D neuronal morphology from light microscopy images. The method uses a prediction and refinement strategy that is based on exploration of local neuron structural features. We extended the rayburst sampling algorithm to a marching fashion, which starts from a single or a few seed points and marches recursively forward along neurite branches to trace and reconstruct the whole tree-like structure. A local radius-related but size-independent hemispherical sampling was used to predict the neurite centerline and detect branches. Iterative rayburst sampling was performed in the orthogonal plane, to refine the centerline location and to estimate the local radius. We implemented the method in a cooperative 3D interactive visualization-assisted system named flNeuronTool. The source code in C++ and the binaries are freely available at http://sourceforge.net/projects/flneurontool/. We validated and evaluated the proposed method using synthetic data and real datasets from the Digital Reconstruction of Axonal and Dendritic Morphology (DIADEM) challenge. Then, flNeuronTool was applied to mouse brain images acquired with the Micro-Optical Sectioning Tomography (MOST) system, to reconstruct single neurons and local neural circuits. The results showed that the system achieves a reasonable balance between fast speed and acceptable accuracy, which is promising for interactive applications in neuronal image analysis.     

The Use of Dimethylsulfoxide for Fixation of Yeasts for Electron Microscopy

Conventional methods of chemical fixation are often inadequate for preserving yeast ultrastructure. The thick cell wall severely limits penetration of fixatives rendering poor detail of the cell wall, membranes, and overall anatomy. Dimethylsulfoxide (DMSO) enhances penetration of chemicals and has been added to fixatives to improve cell preservation. At high concentrations (5 to 50%), however, it affects ultrastructure unpredictably. We found that adding 0.1% DMSO to fixatives greatly improved retention of yeast ultrastructure. Candida albicans, C. glabrata and Aspergillusfumigatus were fixed for 3 hr in 3% paraformaldehyde, 1% glutaraldehyde, 1 mil MgCl₂, 1 mM CaCl₂, 0.1% DMSO in 0.1 M sodium cacodylate buffer followed by 1% OsO₄, 1% K₂Cr₂O₇, 0.85% NaCl. 0.1% DMSO in the same buffer. Thin epoxy sections were post-stained in uranyl acetate and lead citrate. The multilayered character of the cell wall was distinct and well structured. Addition of ruthenium red or alcian blue to the fixatives further enhanced the outer fibrillar layer. The plasma membrane was contiguous and tightly adjacent to the inner manno-protein layer of the cell wall. The cytoplasm was well preserved and the overall preservation of the yeast ultrastructure was significantly improved.     

A Removable Polar Embedding Medium for Light Microscopy

A new plastic embedding medium for light microscopy is described. The monomer mixture consists of equal proportions by volume of acrylonitrile, dimethyl acrylamide and methyl methacrylate, and may be polymerized by exposure to ultraviolet light in the presence of benzoin methyl ether as catalyst. Dithiothreitol may also be added to the monomer mix to limit the degree of polymerization. The resulting polymer is soluble in dimethyl formamide.     

Staining Etched Epoxy Resin Sections for Light Microscopy

Staining of etched sections for light microscopy is described. Azan staining was successful after treatment with potassium dichromate and the use of concentrated dye solutions. To remove osmium for hematoxylin-eosin staining, removal by reduction with ferrocene was used instead of oxidation. Highly selective differentiation after hematoxylin staining was achieved using p-toluenesulfonic acid-DMSO. To enhance eosin staining, a 2-bromoethylamine link between eosin and the tissue was used. Ferrocene also facilitated counterstaining of nuclei with hematoxylin after the PAS reaction. Periodic acid-methenamine silver staining was carried out without modification.     

The Fate of Mitochondria in Ibex-hirus Reconstructed Early Embryos

Nuclear transfer can be used to maintain limited popu-lations of highly endangered species [1–3] . Especiallywhen the oocytes of these species are difficult to obtain,inter-species nuclear transfer seems more appropriate forthis goal. The main methods are as follows: somatic cellis injected directly or fused by electroporation with re-cipient enucleated oocyte. Therefore, nDNA as well asmtDNA ought to be transferred totally or partially to theoocyte. Mitochondria provides adenosine triphos…     

Lead Tetraacetate-Thiocarbohydrazide-Silver Proteinate Method for Light Microscopy of Polysaccharides

Lead tetraacetate-thiocarbohydrazide-silver proteinate reaction sequence for light microscopy of polysaccharides was evaluated on Carnoy's fixed rat liver sections. The results of this evaluation suggest that, on the light microscopic level, the lead tetraacetate-thiocarbohydrazide-silver proteinate method may serve as a practical and histochemically specific alternative to the lead tetraacetate-Schiff reaction for the localization of tissue carbohydrates.     

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.     

Lead Tetraacetate-Thiocarbohydrazide-Silver Proteinate Method for Light Microscopy of Polysaccharides

Lead tetraacetate-thiocarbohydrazide-silver proteinate reaction sequence for light microscopy of polysaccharides was evaluated on Carnoy's fixed rat liver sections. The results of this evaluation suggest that, on the light microscopic level, the lead tetraacetate-thiocarbohydrazide-silver proteinate method may serve as a practical and histochemically specific alternative to the lead tetraacetate-Schiff reaction for the localization of tissue carbohydrates.     

Evaluation of Excitation Light Sources for Incident Immunofluorescence Microscopy

A variety of fluorescent excitation light sources were compared using a standard fluorescein solution or a bacterial conjugate with immunofluorescent microscopy. Quantitative data were obtained with microscope photometric apparatus. Both the quantitative data and comparative conjugate titering suggest that the 450-W xenon arc excited significantly more fluorescence than did the more commonly used 250-W mercury arc or the 100-W halogen lamp. The conjugate could be diluted 4 to 32 times more using the 450-W xenon. Additional advantages of 450-W xenon excitation include sufficient energy of wavelengths between 470 to 490 mm, thus permitting narrow-band excitation resulting in less autofluorescence and the ability to perform fluorescent-antibody procedures without the darkening of ambient room light.     

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos¹, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media². In addition, they grow optimally and quickly at 21-24°C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)³. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher''s choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.Download video file.^{(106M, mp4)}