Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

Morphological and functional aspects of volatile-producing glands in bees (Hymenoptera: Apidae)

Abstract In this paper we focus on the occurrence and morphological aspects of exocrine glands in several bee species. Morphology of head labial, mandibular, Dufour, and abdominal tegumentar glands was investigated under light microscopy, scanning electron microscopy and transmission electron microscopy. Most of such glands present cells with cytoplasm homogeneous and acidophilic, or contain small apparently empty vacuoles. The cytoplasm cells' ultrastructure showed a well developed smooth endoplasmic reticulum, many polymorphic mitochondria, rare Golgi, lipid droplets, myelin figures, and many basal and apical plasma membrane infoldings. All these results are discussed in the text.     

Cymbella Agardh属(硅藻门Bacillariophyta)的一个新化石种的发现及其微细构造

新化石种Cymbella taicuonensis Li et Zheng sp.nov.是在研究西藏西北部日土县台错古湖剖面沉积物中硅藻植物群时发现的,剖面沉积物中保存了非常丰富的硅藻化石,Cymbella taicuonensis就是这些硅藻化石中的一个新化石种。对标本采用光学镜(LM)和扫描电镜(SEM)观察和照相,并对其微细构造进行深入的研究。     

Fluorophores for single-molecule localization microscopy

Super-resolution fluorescence microscopy allows for obtaining images with a resolution of 10–20 nm, far exceeding the diffraction limit of conventional optical microscopy (200–350 nm), and provides an opportunity to study in detail the subcellular structures and individual proteins in both living and fixed cells. Among these methods, single-molecule localization microscopy (SMLM) has become widespread. SMLM techniques are based on special fluorophores capable of photoswitching. The paper presents a classification of such fluorophores and describes their photoswitching mechanisms and successful practical applications. We discuss recent progress and prospects for the development of new effective labels suitable for SMLM.     

低温电镜技术及其在植物（动物）材料研究中的应用

通过一些实例介绍了高压冷冻,冷冻置换和冷冻超薄切片等低温电镜样品制备技术,并且与传统方法对照,说明低温电镜技术的优越性,其中,发菜（Nostoc flagelliforme)营养细胞的冷冻超薄切片（未经化学固定,脱水）所显示的超微结构更客观地反映了生物样品的自然生理状态。此外,应用高压冷冻和冷冻置换的免疫标记电镜技术,首次对发菜营养细胞中的DNA进行定位,明确了核区的位置及范围。     

SPI-EM: towards a tool for predicting CATH superfamilies in 3D-EM maps

In this paper the theoretical framework used to build a superfamily probability in electron microscopy (SPI-EM) is presented. SPI-EM is a new tool for determining the homologous superfamily to which a protein domain belongs looking at its three-dimensional electron microscopy map. The homologous superfamily is assigned according to the domain-architecture database CATH. Our method follows a probabilistic approach applied to the results of fitting protein domains into maps of proteins and the computation of local cross-correlation coefficient measures. The method has been tested and its usefulness proven with isolated domains at a resolution of 8 A and 12 A. Results obtained with simulated and experimental data at 10 A suggest that it is also feasible to detect the correct superfamily of the domains when dealing with electron microscopy maps containing multi-domain proteins. The inherent difficulties and limitations that multi-domain proteins impose are discussed. Our procedure is complementary to other techniques existing in the field to detect structural elements in electron microscopy maps like alpha-helices and beta-sheets. Based on the proposed methodology, a database of relevant distributions is being built to serve the community.     

Imaging cerebroside-rich domains for phase and shape characterization in binary and ternary mixtures

The objective of this paper is to review phase behavior and shape characterization of cerebroside-rich domains in binary and ternary lipid bilayers, as obtained by microscopy techniques. These lipid mixtures provide a format to examine molecular (e.g. headgroup, tail unsaturation, and tail hydroxylation) and thermodynamic (e.g. temperature and mole percentages) factors that determine phase behavior, molecular partitioning, crystal/atomic scale structure, and microstructure/shape (particularly of phase-separated domains). Microscopy can provide excellent spatial (often with high resolution) characterization of cerebroside-rich domains (and their surroundings) to identify, describe or infer with high certainty these characteristics. In the introduction to this review we review briefly the molecular structure, phase behavior, and intermolecular interactions of cerebrosides, in comparison to ceramides and sphingomyelins and in some binary and biological systems. The bulk of the review is then devoted to microscopy investigations of cerebroside-rich domain microstructure and shape dynamics in binary and ternary (one component is cholesterol) systems. Quantitative and/or high-resolution microscopy techniques have been used to interrogate cerebroside-rich domains such as freeze-fracture electron microscopy, atomic force microscopy, imaging elipsometry, two-photon fluorescence microscopy, and LAURDAN generalized polarization in addition to the laboratory workhorse technique of epifluorescence microscopy that allows a quick often qualitative assessment of microstructure and dynamics. We particularly focus on the information these microscopy investigations have revealed with respect to phase behavior, cholesterol partitioning, domain shape, and determinants of domain shape.     

Laboratory cryo soft X-ray microscopy

Lens-based water-window X-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their near-native state with unprecedented contrast and resolution. Cryofixation is essential to avoid radiation damage to the sample. Present cryo X-ray microscopes rely on synchrotron radiation sources, thereby limiting the accessibility for a wider community of biologists. In the present paper we demonstrate water-window cryo X-ray microscopy with a laboratory-source-based arrangement. The microscope relies on a λ=2.48-nm liquid-jet high-brightness laser-plasma source, normal-incidence multilayer condenser optics, 30-nm zone-plate optics, and a cryo sample chamber. We demonstrate 2D imaging of test patterns, and intact unstained yeast, protozoan parasites and mammalian cells. Overview 3D information is obtained by stereo imaging while complete 3D microscopy is provided by full tomographic reconstruction. The laboratory microscope image quality approaches that of the synchrotron microscopes, but with longer exposure times. The experimental image quality is analyzed from a numerical wave-propagation model of the imaging system and a path to reach synchrotron-like exposure times in laboratory microscopy is outlined.     

Description and life cycle of a new Physarum (Myxomycetes) from the Atacama Desert in Chile

A new species of Physarum (Myxomycetes), Physarum atacamense is described in this paper, and details are provided on its life cycle as observed in spore-to-spore culture in agar. The new species was collected during studies of the Atacama Desert in Chile. It has been collected directly in the field and isolated in moist chamber cultures prepared with material from an endemic cactus. The combination of characters that make this species unique in the genus are its large fusiform nodes of the capillitium, its long, bicolored stalk and the very dark brown and densely warted angular spores. The morphology of specimens of this myxomycete was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details and life cycle stages are included in this paper. The importance of resistant stages in the life cycle of this myxomycete is stressed, and the close association of this myxomycete with its plant substrates is discussed.     

湖北安陆新的恐龙蛋类型的发现及其意义

本文记述的恐龙蛋化石标本,采自湖北省江汉盆地公安寨组下部.蛋化石保存完好.在普通光学显微镜和扫描电镜下观察,蛋壳的显微结构完整,清晰可见.根据钙质蛋壳基本结构单位的形态及排列特征,笔者建立了—新属,新种——Dendroolithus wangdianensis gen. et sp. nov.,代表恐龙蛋类一个新科——Dendroolithidae fam. nov.此外,残存的卵壳膜纤维化石的发现,将为进一步研究恐龙蛋卵壳膜的结构和氨基酸组成提供宝贵材料.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Optical coherence hyperspectral microscopy with a single supercontinuum light source

In the paper, we have developed an optical coherence hyperspectral microscopy with a single supercontinuum light source. The microscopy consists of optical coherence tomography (OCT) and hyperspectral imaging (HSI), which can visualize the structural and functional characteristics of biological tissues. The 500 to 700 nm band is selected for HSI and OCT imaging, where HSI enables imaging of oxygen saturation and hemoglobin (Hb) content, while OCT acquires structural characteristics to assess the morphology of biological tissues. The system performance of the optical coherence hyperspectral microscopy is verified by normal mice ears, and the practical applications of the microscopy is further performed in 4T1 and inflammation Balb/c mice ears in vivo. The experimental results demonstrate that the microscopy has potential to provide complementary information for clinical applications.     

泰山虫草子实体形态特征与rDNA变异位点分析

用光学显微镜和扫描电镜对采自泰山的虫草子实体可孕部分进行形态观察,结果表明泰山虫草在子实体形态、子囊的排列方式、子囊的包埋方式、子囊壳的形态等与九州虫草完全一致。对虫草核糖体DNA的18S、28S、ITS区克隆测序,并进行序列比对后发现两种虫草的碱基差异较小,其同源性高达99%,符合种内遗传特性。结合形态学和分子生物学两方面的特征,初步推断两者可能都是九州虫草。     

Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque

This paper quantitatively defines the nanoscale topography of the basement membrane underlying the anterior corneal epithelium of the macaque. Excised corneal buttons from macaques were placed in 2.5 mM ethylenediaminetetraacetate (EDTA) for 2.5 h, after which the epithelium was carefully removed to expose the underlying basement membrane. The integrity of the remaining basement membrane was verified using fluorescent microscopy in conjunction with antibody staining directed against laminin and collagen type IV as well as transmission electron microscopy. Characterization of the surface of the basement membrane was performed using transmission electron microscopy, high-resolution, low-voltage scanning electron microscopy, and atomic force microscopy. Quantitative data were obtained with all three imaging techniques and compared. The basement membrane has a complex topography consisting of tightly cross-linked fibers intermingled with pores. The mean elevation of features measured by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy was 149 +/- 60 nm, 191 +/- 72 nm, and 147 +/- 73 nm, respectively. Mean fiber diameter as measured by SEM was 77 +/- 44 nm and pore diameter was 72 +/- 40 nm, with pores occupying approximately 15% of the total surface area. Similar feature types and dimensions were also found for Matrigel, a commercially available basement membrane-like complex, supporting that a minimum of artifact was introduced by corneal preparative procedures to remove the overlying epithelium. Topographic features amplified the surface area over which cell-substratum interactions occur by an estimated 400%. The three-dimensional structure of the basement membrane exhibits a rich complex topography of individual features, consisting of pores and fibers with dimensions ranging from 30 to 400 nm. These nanoscale substratum features may modulate fundamental cell behaviors such as adhesion, migration, proliferation, and differentiation.     

Fiber analysis of an insect peripheral nerve: Light vs. electron microscopy

This paper describes the morphology of one of the two major femoral nerve trunks (nerve V of Dresden and Nijenhuis (1958)) of the cockroach Blaberus discoidalis as revealed by light microscopy, scanning electron microscopy and transmission electron microscopy. Fiber analysis of light and electron micrographs of thick and thin sections taken from adjacent areas of the same nerve yield significantly different data about the number and size distribution of the component axons of nerve V. The light microscopic image indicates one example of nerve V contains 2030 individual axons with a mean diameter of 1.4 μ; the electron image indicates the nerve contains 5794 axons with a mean diameter of 0.56 μ. The disparity between the two types of data is due to the large number of very small axons (0.2 μ or less in diameter) that course through the femoral nerve.     

双菱藻科（硅藻门）——四个中国新纪录种

本文报道了采自新疆及云南地区的双菱藻科（硅藻门）中国新纪录植物4种,分别为耳蜗波缘藻Cymato—pleura cochlea Brun、维苏双菱藻Surirella visurgis Hustedt、盾状马鞍藻Campylodiscus clypeus Ehrenberg和莱温德马鞍藻Campylodiscus levanderi Hustedt。通过光镜（LM）和扫描电镜（SEM）观察,对其分类学特征进行了详细的描述,并介绍了他们的生境特征等。     

Spore to spore culture of Didymium operculatum, a new Myxomycete from the Atacama Desert of Chile

A new species of Didymium (Myxomycetes), D. operculatum, is described in this paper, and details of its life cycle are provided. The new species was recorded during studies of the Atacama Desert in Chile. It has been collected directly in the field and isolated in moist chamber cultures prepared with material from an endemic cactus. The distinguishing characters of this species are its dehiscence by means of an apical operculum combined with a whitish calcareous stalk and the banded reticulate ornamentation of the spores. The morphology of this new myxomycete was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included in this paper. Some comments are made on the patterns of distribution of Didymium species in arid lands and adaptive characters enabling this genus to colonize such extreme environments. It is proposed that a longer cycle and the ability to resort to resistant forms many times during their development reflect the response of these myxomycetes to the largely unfavorable conditions of their environment.     

FITC-Dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy,fluorescence light microscopy and electron microscopy

Summary Coupling fluorescein-isothiocyanate to dextrans (FITC-D) extends the usefulness of the dextrans as electron microscopic tracer particles by permitting preceding fluorescence stereo microscopy and high-power light microscopy of the tissue specimens. The fate of the tracer may thus be studied in vivo during the experiment, during fixation, and during the succeeding tissue processing. A study of some simple physicochemical characteristics of the tracer, and the influence, if any, of the fixing agent are also made possible. FITC-D was found to be uncharged in the pH range from 6.5 to 8.5, more rapidly precipitated by acetone than by alcohol, and to react with glutaraldehyde and osmium tetroxide in an unknown way during tissue fixation. FITC-D with molecular weights 70,000 and 150,000 showed no signs of diffusion during tissue preparation with the methods reported in the paper, whereas FITC-D 40,000 did so to a slight degree, when the tissue was kept for several days in the fixative vehicle. Securing the preservation of the lower molecular weight FITC-Ds during tissue fixation and preparation is more difficult and the described methods are not adequate. Dextrans provoke an anaphylactic reaction in most rat strains, but are well tolerated by Wistar Furth rats. The introduction of FITC into the dextran molecule might alter the biological reactions, but was also well tolerated by Wistar Furth rats. Combined fluorescence stereo microscopy, fluorescence microscopy of sections, light microscopy of stained sections and electron microscopy made it possible to follow a particular microcirculatory area, selected in vivo, to the final study in the electron microscope.     

激光扫描共聚焦显微镜在蛋白质晶体生长研究中的应用

激光扫描共聚焦显微镜与普通光学显微镜相比,其分辨率高,同时具有可对样品进行非侵入性无损伤断层扫描,以及对样品形貌进行三维成建等特点,因此,可作为研究晶体生长强有利的工具。本文介绍了其在定量测量晶体的个数,重组三维图像以获得晶体生长的过程信息及测定晶体生长台阶动态变化等方面的应用。还对激光扫描共聚焦显微镜在晶体生长研究的其它方面应用前景作了展望。     

FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy

Coupling fluorescein-isothiocyanate to dextrans (FITC-D) extends the usefulness of the dextrans as electron microscopic tracer particles by permitting preceding fluorescence stereo microscopy and high-power light microscopy of the tissue specimens. The fate of the tracer may thus be studied in vivo during the experiment, during fixation, and during the succeeding tissue processing. A study of some simple physicochemical characteristics of the tracer, and the influence, if any, of the fixing agent are also made possible. FITC-D was found to be uncharged in the pH range from 6.5 to 8.5, more rapidly precipitated by acetone than by alcohol, and to react with glutaraldehyde and osmium tetroxide in an unknown way during tissue fixation. FITC-D with molecular weights 70,000 and 150,000 showed no signs of diffusion during tissue preparation with the methods reported in the paper, whereas FITC-D 40,000 did so to a slight degree, when the tissue was kept for several days in the fixative vehicle. Securing the preservation of the lower molecular weight FITC-Ds during tissue fixation and preparation is more difficult and the described methods are not adequate. Dextrans provoke an anaphylactic reaction in most rat strains, but are well tolerated by Wistar Furth rats. The introduction of FITC into the dextran molecule might alter the biological reactions, but was also well tolerated by Wistar Furth rats. Combined fluorescence stereo microscopy, fluorescence microscopy of sections, light microscopy of strained sections and electron microscopy made it possible to follow a particular microcirculatory area, selected in vivo, to the final study in the electron microscope.