X射线对我国两种典型土壤中微生物活性及群落结构的影响

【目的】X射线断层扫描技术(X-ray micro-Computed Tomography,micro-CT)能够原位、无损伤的解析土壤物理结构,有望与土壤微生物研究结合,以有助于更好的了解土壤生态系统。由于土壤的高度异质性,X射线扫描和土壤微生物分析应为同一样品,但是关于X射线扫描后的土壤样品是否兼容土壤微生物分析却鲜有报道,即X射线扫描是否影响土壤微生物的活性及群落尚未明确。【方法】本研究采集我国华北地区潮土和亚热带红壤,利用平板计数、微量热技术和高通量测序技术研究了X射线扫描对可培养微生物数量、土壤微生物的代谢热和群落结构的影响。【结果】X射线辐射显著降低了2种土壤中活体细菌的数量,同时微生物的代谢活性也发生改变;在分子水平上,基于细菌16S r RNA基因的高通量数据显示2种土壤的细菌多样性指数发生了变化,而其群落结构均无改变。【结论】X射线断层扫描技术并不兼容土壤微生物功能的研究;但可兼容基于分子生物学的微生物群落结构分析。     

XST-122型调制反差显微镜

调制反差显微镜是中国科学院上海细胞生物学研究所研制成功的一种新型显微成象系统,于1984年10月在上海通过鉴定。现由中国科学院上海细胞生物学研究所和宁波光学仪器厂联合生产,将于1985年5月投放市场。该显微镜除了具有一般亮视野显微镜的性能外,更主要的是它能观察和拍摄透明的样品、未经染色的活细胞和组织切片。与相差显微镜相比较,它能产生     

东海表层沉积物纯培养与非培养细菌多样性

于2011年7月采集了浙江舟山沿岸海底沉积物样品(122°10′41″E,29°49′7″N),通过埋片原位观察、荧光显微计数、纯培养菌分离以及非培养细菌构建克隆文库的方法,分析和研究了东海表层沉积物细菌群落结构及其多样性.埋片原位观察和荧光显微计数法的结果表明:沉积物样品中细菌的细胞数量为(9.30±3.44)×107 cells/g;通过分离纯化共获得313株细菌,分属于20个属,4种培养基对细菌的分离效果依次为RO＞M l＞Zobell 2216＞MR2A;常规形态学与生理生化鉴定结果显示芽孢杆菌属(Bacillus)和海球菌属(Marinococcus)从数量和种类上皆为优势菌属,分别占分离菌株总数的21.08％和17.25％;对73株典型海洋细菌进行16S rDNA分子鉴定发现,厚壁菌门(57.5％)、γ-变形杆菌纲(32.9％)、黄杆菌纲(4.1％)和放线菌纲(5.5％)等为主要类群.非培养细菌克隆文库序列分析发现:细菌克隆子主要属于厚壁菌门和变形杆菌门,而芽孢杆菌纲和γ-变形杆菌纲是上述两个门的优势类群.综合纯培养与非培养数据得出东海海域表层沉积物细菌多样性丰富,具有进一步开发研究的价值.     

甲醛固定对Live/Dead BacLight Bacterial Viability Kit死活细菌染液荧光显微计数海洋细菌的影响

利用Live/Dead BacLight Bacterial Viability Kit 死活细菌染液对采自湛江东海大堤海水、沉积物细菌和大型海藻拟刚毛藻（Cladophoropsis zollingeri）内生细菌数量进行了甲醛固定处理前后的荧光显微计数对比分析。结果表明,新鲜样品（不加甲醛固定）、甲醛刚固定样品、甲醛固定1 周样品和甲醛固定2 周样品中海洋细菌数量差异不显著（p>0.05）。甲醛固定对Live/DeadBacLight Bacterial Viability Kit 死活细菌染液荧光显微计数海洋细菌数量无显著影响,固定后的样品可在2 周内完成计数。     

白皮松针叶内皮层在盐胁迫中的屏障作用研究

应用荧光显微技术、傅里叶变换显微红外光谱分析(FTIR)、扫描电镜及X-射线能谱微区分析等手段,对白皮松(Pinus bungeana)子叶、初生叶及2a生针叶内皮层细胞径向壁的显微结构特征、化学成分,以及在叶子横切面上Na和Cl的微区分布进行分析。通过荧光显微观察发现,白皮松子叶内皮层不具凯氏带,而初生叶及2a生针叶均存在凯氏带加厚现象。根据FTIR的检测结果显示:子叶内皮层细胞径向壁不含木栓质或极少,2a生针叶内皮层细胞径向壁木栓质含量高于初生叶。对相应区域的X射线微区分析表明,子叶内皮层对Na和Cl在质外体运输中不起障碍作用,而初生叶与2a生针叶内皮层阻碍Na和Cl以质外体途径进入维管组织。研究结果表明:具凯氏带加厚的内皮层细胞壁中木栓质含量决定其在质外体运输过程中的生理功能。     

定量电子束显微分析中生物薄样品的质量损失

对多种生物薄样品和标样进行电子探针X射线能谱显微定量分析,分别以电子束轰击后样品的O Kα峰计数和介于4.2-6.2keV区间的连续X－射线计数变化监测质量损失,结果显示样品O Kα峰计数减少幅度大于连续X－射线计数减少幅度,在相同的分析条件下,各样品质量损失程度不相同（P＜0.05）。培养肝癌细胞冷冻干燥超薄切片、明胶冷冻干燥超薄切片、BSA薄膜、氨基塑料超薄切片、红细胞冷冻干燥超薄切片和卵黄高磷蛋白薄膜样品的质量损失分别为33％、28％、26％、18％、13％和13％,以上结果提示：以O Kα峰计数的减少监测样品的质量损失较敏感,在进行生物薄试样定量EPMA时应对各样品的质量损失进行相应校正。     

新型快速显微多道分光光度系统及其应用

介绍一种新型快速显微多道分光光度系统。利用倒置显微镜、衍射光栅、线阵CCD等构成的显微多道分光光度系统,可对微区样品在350nm-800nm（波长分辨率为0．2nm)光谱波长范围内进行快速光谱检测,最快检测时间为1ms。利用该系统进行的人血红细胞内血红蛋白测量分析表明,该具有高灵敏度、快速、无扰、在位、多光谱显微测量的特点,在生命科学研究中动态监测细胞生命过程的变化。     

低剂量超软X射线对染色体的损伤效应

用~(60)Coγ-射线或深部治疗的X光机的“硬”X射线照射微生物及动植物,引起细胞染色体的突变,并用于育种工作,这已成为常规的育种手段。有关硬X射线引起的植物细胞染色体的辐射损伤效应,报道甚多,而低剂量的X射线,尤其是超软X射线的辐射染色体损伤效应,报道却寥寥无几。本工作利用上海新跃仪表厂生产的DGX-4型软X射线机,钼靶、波长为0.62—0.71的X射线(根据这一波段范围应属     

大豆属植物茎的次生木质部结构研究

利用光学显微技术和扫描电镜技术,对大豆属Clycine L.的4种类型植物茎材进行比较解剖学研究.结果表明,4种类型植物的茎材横切面均为散孔材。野生大豆茎材多为单管孔.少见复管孔;半野生大豆具复管孔,少见多细胞的管孔链;半栽培大豆茎材中复管孔和管孔链较多;而栽培大豆的复管孔和管孔链更多且普遍.野生大豆单列射线多,多列射线少半野生大豆有少数多列射线;半栽培大豆多列射线较多;栽培大豆多列射线细胞组成的射线最多.大豆种植物的次生木质部导管的侵填体分布不同,各种结构的演化途径为野生大豆→半野生大豆→半栽培大豆→栽培大豆野生大豆结构较原始,栽培大豆最进化.     

显微分光光度法测定Co~(60)辐照后大白鼠外周淋巴细胞核内DNA含量变化

显微分光光度法是利用分光光度法的原理,以一定波长的单色光在显微镜下对生物样品微细结构中的化学物质进行定量测定。显微分光光度法测定的不是一个细胞,而是一个细胞群体内的某种物质(如DNA),其中各个细胞内的物质含量不完全相同,然而,一个细胞群体的某物质含量有一定的分布,通常用组织图来表示,正常细胞胞核的DNA含量只与染色体的数目有关,如呈规律性的成倍增加,即为2倍体、4倍体等。用这种方法,可以鉴定正常细胞受到某种外界环境刺激后所发生的变化,如癌变等。我们采用这种技术探测大白鼠受Co~(60)γ射线照射后外周血淋巴细胞核内DNA含量的变化。一、材料和方法选择体重为150g—200g的雄性大白鼠,每16—20只为一批,进行Co~(60)r照射,剂量     

Soft x-ray microscopy

Soft x-ray microscopes are beginning to provide information to complement that obtained from optical and electron microscopy. Soft x-ray microscopy can deliver 30-nm resolution images of hydrated cells up to approximately 10 microns thick, and efforts towards obtaining higher resolution are under way. Although living specimens cannot be studied readily except in single exposures, fixed samples can be imaged at high resolution, and flash-frozen specimens can be studied without chemical modification and without significant radiation damage. Tomography is being developed for 3-D imaging, and spectromicroscopy offers unique capabilities for biochemical mapping of unlabelled structures beyond those of gold and fluorescent labels. Currently, most soft x-ray microscopes operate at synchrotron radiation facilities, but laboratory-scale microscopes are being developed too.     

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological “nanomachines” it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 μm in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.     

Ultraviolet Germicidal Irradiation and Its Effects on Elemental Distributions in Mouse Embryonic Fibroblast Cells in X-Ray Fluorescence Microanalysis

Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologically important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results.     

3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography

The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.     

Characterization of the performance of a 200-kV field emission gun for cryo-electron microscopy of biological molecules

The value of an electron microscope equipped with a field emission gun (FEG) was first revealed in materials science applications. More recently, the FEG has played a crucial role in breaking the 10A barrier in single-particle reconstructions of frozen hydrated biological molecules. The standard high-resolution performance tests for electron microscopes are made close to focus, at several hundreds of A underfocus at a magnification of 500,000x or more. While this is appropriate for materials science specimens, it is not suitable for observing frozen hydrated biological specimens with which the optimum underfocus is of the order of 1 micron or so and the magnification is limited by radiation damage to roughly 30,000 to 60,000x. Thus, in order to access the performance of a cryo-electron microscope for high-resolution 3D electron microscopy of biological molecules, additional tests are necessary. We present here resolution tests of a 200-kV FEG using frozen hydrated virus suspensions. The extent and amplitude of the contrast transfer function are used as a test of the performance. We propose that small spherical viruses close to 300A in diameter, such as the picornaviruses or phages, make good specimens for testing the performance of an electron microscope in cryo-mode.     

Structure and composition of ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells

Ferritin cores isolated from human spleen, limpet (Patella vulgata) hemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated by high resolution transmission electron microscopy, electron diffraction and chemical analysis. Hemosiderin particles isolated from thalassemic spleens also have been studied. The results show that there is a marked difference in structure and composition of the biomineral phases. Human ferritin and hemosiderin particles are single domain crystals of hydrated iron (III) oxide (ferrihydrite). Lattice fringes were low in contrast and often discontinuous within the central regions of the core. Heat treatment of human ferritins results in a 5 A shrinkage in particle size and an increase in the single crystalline nature of the core. In contrast, lattice images and electron diffraction of limpet and bacterial cores show no evidence of long-range crystallographic order. Chemical analysis indicates a high inorganic phosphate (Pi) (Fe/Pi = 1.71) content in bacterial ferritin compared with human ferritin (thalassemic) (Fe/Pi = 21.0). The high Pi content of bacterial ferritin suggests a hydrated amorphous iron (III) phosphate mineral core. Structural disorder within the limpet and bacterial cores may be associated with increased Pi content and increased oxidation in Fe(II), resulting in rapid mineral deposition. Growth of the iron (III) oxide cores in human ferritin is discussed on the basis of high resolution electron microscopy results.     

Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ

AIMS: To investigate the use of confocal Raman microspectroscopy (CRM) for the analysis of the structure, composition and development of fully hydrated biofilms. METHODS AND RESULTS: Pseudomonas aeruginosa PAO1 biofilms were cultured in a flow cell in minimal nutrient medium (artificial sea water) and their development was followed for up to 3 weeks. The spectroscopic signature of the biofilm cells and extracellular polymeric substances (EPS) were differentiated and their distribution in biofilm colonies and within water channels was mapped in-plane and -depth. The colonies were initially amorphous, mainly composed of cells with no detectable amount of EPS. They developed rapidly to give round colonies composed of a cellular core enclosed in a sheath of EPS. The EPS continued to increase and spread throughout the biofilm to become the dominating feature of aged colonies. Colonies with a liquid core morphology - characteristic of the seeding dispersal process - were also observed. CONCLUSIONS: This study demonstrated that CRM can be used to monitor the distribution of biofilm components in fully hydrated undisturbed biofilms over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Confocal Raman microspectroscopy facilitates the analysis of hydrated, live bacterial biofilms as a function of space and time, thus making it a suitable technique for investigating the effects of various additives and environmental factors on biofilm growth.     

Schistosoma manonsi: cercarial penetration of host epidermis at the ultrastructural level

Invasion of the outer layers of the epidermis of mouse ear skin by cercariae of Schistosoma mansoni within 7 min of their application to it has been studied with the optical and the scanning and transmission electron microscopes.Entrance of cercariae was under the edges of the dead flattened keratinized cells of the horny layer (squames), and penetration through this layer was by disarticulation of the stacks of squames at their interdigitations. Mucus from the postacetabular glands was recognized with the light and electron microscopes on the skin surface, especially at squame edges and between layers of squames and along the keratogenous zone. The findings suggested that disarticulation of the squames was not effected solely by the muscular probing and pushing of the parasite, but that it might be aided by swelling of the mucus secretion deposited in the area from the postacetabular glands. Loosening of the interlocked edges of the squames by enzymatic action is also a possibility, but was not evaluated for this report.The migration path along the keratongenous zone was marked by extensive damage to the transitional cells of the granular layer subjacent to the squames. Packets of secretion from the cercarial preacetabular glands were identified below the horny layer in the cytoplasm of these cells. It was considered that the host tissue damage in this area was the result not only of tearing of the tissues by passage of the spiny schistosomules, but also of enzymatic activity, the enzyme source being the granules in the packets of preacetabular gland secretion.     

FISH and chips: marine bacterial communities analyzed by flow cytometry based on microfluidics

To unveil the structure of natural marine pelagic bacterial communities, PCR-based techniques as well as fluorescence in situ hybridizations (FISH) were successfully performed in the past. Using fluorescence microscopes or confocal laser scanning microscopes (CLSM) for the analysis of FISH experiments, it was possible to differentiate bacterial communities, but most attempts to combine flow cytometry and FISH for this purpose have failed till now. Here we present a successful analysis of FISH experiments of natural marine pelagic bacterial communities using a flow cytometer based on microfluidics (Agilent 2100 bioanalyzer). Marine water samples were enriched on polycarbonate filters and hybridized with Cy5 labeled gene probes of different phylogenetic depth. Bacteria were detached from the filters and subsequently analyzed in the Cell Chip of the Agilent 2100 Bioanalyzer. Samples were counter-stained using SYTOX. In all samples the EUB338 positive signals could be clearly differentiated from those of the NON probe. Furthermore a dominance of alpha-protebacteria (as indicated by the probes ALF968 and G rB) could be observed. Microfluidics based flow cytometry is a promising technique for the analysis of natural bacterial communities from the marine environment.     

Ultrastructure of the in situ adherence of Mobiluncus to vaginal epithelial cells

From patients with bacterial vaginosis motile, anaerobic, comma-shaped bacteria can be isolated, which have recently been placed into the new genus Mobiluncus. In this study, electron microscopy was used to examine the in situ adherence of these motile curved rods to detached epithelial cells (comma cells) in vaginal fluid from two patients with bacterial vaginosis. Thin sections showed that the curved rods attached both directly to the epithelial cell surface and at various distances from it. It is concluded that after initial attachment these motile bacteria can grow at the epithelial cell surface in sessile microcolonies. Ruthenium red staining demonstrated a coating of precipitated glycocalyx material both on the surface of the curved rods and on their flagella. This may indicate that in situ the adherent curved rods were enclosed in a very hydrated matrix of exopolysaccharides. Conspicuous was the ability of the curved rods to attach to the epithelial cell surface via their cell tips. However, in situ no specialized bacteria cell surface structures were seen that might explain this polar attachment. Electron microscopy of pure cultures demonstrated that both Mobiluncus curtisii subsp. curtisii and Mobiluncus mulieris can produce a glycocalyx in vitro.