油杉小孢子发生的激光扫描共聚焦显微镜观察

应用激光扫描共聚焦显微镜研究了油杉小孢子的发生过程。油杉样品经曙红-Hoechst 33342双染和冬青油透明后,分别以UV、488 nm激光进行单扫描或系列扫描,并对系列扫描图像进行三维重建。观察结果表明,LSCM图像更能清晰地展示油杉花粉母细胞减数分裂的过程。粗线期染色体密集并缠绕成一团,占据核的一部分,核仁靠在这一团染色体的一边;减数分裂存在扩散双线期;在减数分裂过程中胞质分裂为同时型。观察到中期I、Ⅱ形成的纺锤体和四分体时期胼胝质壁的沉积等。研究结果表明,曙红-Hoechst 33342双染法可用于花粉发育阶段的鉴定,LSCM是研究花粉生物学的理想工具。研究为探究其濒危机制提供有意义资料,为其保护生物学和系统分类学提供胚胎学依据。     

水稻双受精过程的共聚焦显微镜观察

(郑州大学离子束生物工程省重点实验室,郑州450052)摘要:首次利用核特异荧光染色与整体透明技术并利用激光扫描共聚焦显微镜(LSCM)对水稻双受精过程进行了观察。激光扫描共聚焦显微镜具有"组织与细胞CT"的功能,可以对整体组织进行扫描并构建三维结构。经荧光染色及透明处理后,在激光扫描共聚焦显微镜下经488nm激光激发,胚囊内各细胞的细胞核以及核仁发出明亮荧光,细胞核轮廓比较清晰,层次感强,并能清晰观察到胚囊内各细胞的结构特点和空间位置关系。不论是对开花前较小的成熟胚囊材料还是开花后较大的胚囊材料都取得了较好的观察效果。同传统的光学显微镜和荧光显微镜相比,其观察到的图像更清晰、更直观、更具立体感。     

共聚焦激光扫描对呼吸窘迫综合症大鼠肺组织的观察

目的：为了更直观地观察和显示呼吸窘迫综合症(acute respiratory distress syndrom,ARDS)典型的病理变化(肺泡内形成一层蛋白质透明膜)。方法：利用百草枯(Paraqual)染毒SD大鼠复制ARDS实验动物模型,取肺病理组织,切片,试剂Goat—Anti-Rat—FITC IgM IgG染色,共聚焦激光扫描显微镜(confocal laser scarming microscope,CLSM)观察。结果：CLSM能清晰到样品内不同层面的病理变化。结论：共聚焦激光扫描显微镜能清晰观察样品内不同层面的结构,相比于传统的光学显微镜,其观察到的图像更直观、更具立体感,能更好表达ARDS的病理变化特征。     

激光共聚焦显微镜与光学显微镜之比较

激光扫描共聚焦显微镜在活细胞的动态检测、光学切片和三维结构重建等方面较光学显微镜有质的飞跃。本文对激光扫描共聚焦显微镜和光学显微镜进行了比较和讨论,并简单介绍多光子激光扫描显微镜。     

激光扫描共聚焦显微镜在植物学中的应用

激光扫描共聚焦显微镜(LSCM)是普通光学显微镜与激光和计算机及其相应的软件技术组合的产物,实现了连续光学切片,能在亚细胞水平观察细胞骨架的动态变化、细胞内特异蛋白、钙等离子的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。并广泛应用于生物三维结构重组及动态分析。本文综述了应用激光扫描共聚焦显微镜技术在植物细胞学、植物发育、组织化学以及基因表达、检测等领域取得的进展。     

利用激光扫描共聚焦显微镜研究植物细胞发育形态学变化

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究植物细胞发育形态学变化。结果表明,利用紫外激光(351 nm)扫描可以清楚地观察到拟南芥叶片表皮细胞的形态及其变化,在已分化的叶片表皮上可观察到包括“铺垫”表皮细胞(epidermal pavement cells)、气孔保卫细胞(guard cell)、气孔伴胞(subsidiarycells)、表皮毛细胞(trichomes)和表皮毛的足细胞(socket cells)等多种形态不同的细胞种类;利用蓝光激光(488nm)辅助曙红浅染,可清晰地显示出拟南芥根生长区内部的各种原始细胞,包括静止区(quiescent center)细胞、皮层/内皮层原始细胞(cortex/endodermal initial cell)、表皮/根冠原始细胞(epidermal/root cap initial cell)和中柱/根冠原始细胞(columella/root cap initial cell)等。利用双光子激光(800 nm)连续扫描30 s可以诱发叶绿体产生自发荧光,并可观察到叶绿体在叶肉细胞中的运动轨迹。结果说明激光扫描共聚焦显微镜在植物细胞形态及发育研究上具有独特的功能。     

共聚焦荧光镜检术揭示大蒜鳞片细胞间期核中的F肌动蛋白网络

采用免疫荧光和荧光分子探针技术与共聚焦激光扫描显微镜观察相结合,对大蒜（Ａｌｌｉｕｍ ｓａｔｉｖｕｍ Ｌ．）鳞片细胞间期核中是否存在Ｆ肌动蛋白进行了研究。结果表明,以兔抗肌动蛋折抗体为一抗、ＦＴＴＧ－羊抗兔ＩｇＧ抗体为二抗进行免疫荧光标记实验,在荧光镜下观察到蒜瓣薄壁组织的细胞核及表皮细胞核均发出明亮的黄绿色荧光经共聚焦激光扫描显微镜进一步检查,整个细胞核呈黄绿色荧光,说明其中含有肌动蛋白。经ＴＲ     

活体染色显微技术和绿色荧光蛋白在本科实验教学中的应用

激光扫描共聚焦显微镜的原理和使用是本科生细胞生物学实验教学中的重要内容。目前,在细胞生物学实验教学中常使用绿色新鲜的植物叶片作为实验材料,在激光扫描共聚焦显微镜下对叶绿体的自发荧光进行观察。叶绿体的自发荧光信号强而且范围广,使学生难以清晰地理解特异性的荧光信号。该文通过对转p35S::Naa10-GFP基因拟南芥幼苗的根尖进行活体染色[碘化丙啶(propidium iodide,PI)和4′,6-二脒基-2-苯基吲哚(4′,6-diamidino-2-phenylindole,DAPI)染色],在不同的激发光下,收集相应的荧光信号,通过计算机辅助成像,获得不同颜色叠加的特异荧光信号图像。该实验设计简单可行,获得的图像清晰且便于观察,能够使初学者直观并且深刻理解激光扫描共聚焦显微镜的原理和使用方法,适合在高校细胞生物学实验教学中推广,同时也为研究其他蛋白质亚细胞定位提供技术参考。     

利用洋葱鳞叶表皮细胞观察植物细胞结构实验的改进

以洋葱表皮细胞为研究材料,通过中性红液泡染色和质壁分离实验相结合,将能更清晰地观察到细胞壁,细胞质膜,细胞质,液泡膜,液泡,细胞核,核仁等结构,达到光学显微镜下观察植物细胞基本结构的教学目的。改进后的实验大大提高了利用洋葱表皮细胞观察植物细胞结构实验的教学效果,同时可以通过观察不同部位液泡体积变化,了解植物细胞的动态发育,使学生掌握了更多的知识点。     

激光扫描共聚焦显微镜在花粉研究中的应用

激光扫描共聚焦显微镜（Laser scanning confocal microscope,LSCM）是普通光学显微镜、激光、计算机及其相应的软件技术结合的产物,能实现连续光学切片和生物三维结构重组及动态分析.作为一种先进的技术手段,LSCM技术已经为花粉生物学的研究提供了有价值的新资料,大大地推动了植物生殖生物学的发展.本文综述了LSCM技术在花粉粒形态（形状、大小及三维重建）、花粉的内部结构（如细胞骨架）、花粉的遗传学、花粉的发育、花粉的萌发、花粉自发荧光特性、孢粉研究等方面中的应用,并指出了LSCM的不足之处,最后提出了LSCM技术在花粉研究中的应用展望.未来LSCM技术应该与多光子技术、活细胞工作站技术相结合使用,才能更准确地进行花粉动态研究的实时监测.     

Studies of the Mature Pollen of Spinacia oleracea after Freeze Substitution and Observed with Confocal Laser Scanning Fluorescence Microscopy

The three-dimensional configuration of the nuclei of the trinucleate pollen grain of Spinacia oleracea L. has been examined by means of confocal laser scanning fluorescence microscopy (CLSM). It shows the presence of a male germ unit (MGU) in which all three nuclei are usually positioned in the periphery of the pollen grain. After freeze fixation and freeze substitution, the ultrastructure is better preserved than with standard chemical fixation. It shows the presence inside the pollen grain of mitochondria, dictyosomes, large starch-containing plastids, endoplasmic reticulum (ER), vacuoles and the MGU. In the sperm cells mitochondria, vesicles, dictyosomes and ER are also found. No microtubules were found in the grain and only very few inside the sperm cells. This is in contrast with earlier published results where fluorescent-labeled antibodies were used.     

Cytoskeleton in Pollen and Pollen Tubes of Ginkgo biloba L.

The distribution of F-actin and microtubules was investigated in pollen and pollen tubes of Ginkgo biloba L. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. A dense F-actin network was found in hydrated Ginkgo pollen. When Ginkgo pollen was germinating,F-actin mesh was found under the plasma membrane from which the pollen tube would emerge. After pollen germination, F-actin bundles were distributed axially in long pollen tubes of G. biloba. Thick F-actin bundles and network were found in the tip of the Ginkgo pollen tube, which is opposite to the results reported for the pollen tubes of some angiosperms and conifers. In addition, a few circular F-actin bundles were found in Ginkgo pollen tubes. Using immunofluorescence labeling, a dense microtubule network was found in hydrated Ginkgo pollen under confocal microscope. In the Ginkgo pollen tube, the microtubules were distributed along the longitudinal axis and extended to the tip. These results suggest that the cytoskeleton may have an essential role in the germination of Ginkgo pollen and tube growth.     

扇脉杓兰花粉超微结构及花粉管生长的观察

为明确扇脉杓兰花粉形态结构及雄性生殖特性,利用扫描电镜、透射电镜和荧光显微镜对花粉形态和超微结构及花粉管生长过程进行观察。结果表明:(1)扇脉杓兰单粒花粉长球形,表面光滑无特征纹饰,有少量胶黏物质,一些表面有2个或以上的深凹陷,凹陷内有球形突起的内容物。(2)花粉壁分为由棒状的基柱小单元组成的外壁和纤维素果胶组成的内壁,有覆盖层;生殖细胞近圆形,细胞核大而致密;营养细胞多弧形,核质分散;花粉粒细胞质含有大量的线粒体、质体和小泡等细胞器,淀粉、蛋白质和多糖含量丰富。(3)花粉管萌发后沿子房壁方向伸长,授粉20d花粉管伸长生长并不明显,授粉30d伸长的花粉管中出现大量胼胝质塞,并且伸长方向转为胚珠中间,花粉管逐渐接近胚珠,在授粉后50d基本完成受精作用。研究认为,扇脉杓兰花粉发育正常,不阻碍有性生殖过程。     

Changes in volume,surface area,and frequency of nuclear pores on the vegetative nucleus of tobacco pollen in fresh,hydrated, and activated conditions

The vegetative nucleus (VN) of Nicotiana tabacum L. has been qualitatively and quantitatively studied in fresh, hydrated, and activated pollen. Techniques included the use of optical sectioning by confocal scanning laser microscopy to obtain volume and surface area measurements, and stereoscopic pairs; and freeze-etch electron microscopy to estimate the frequency of nuclear pores per m² in the vegetative nucleus. Several morphological changes were observed to occur in pollen grain nuclei during the early processes of tube growth. In freshly dehisced pollen grain, the vegetative and generative nuclei were side by side, but following hydration and activation of the grain, the elongated generative nucleus became partially surrounded by the vegetative nucleus. It was found that during hydration, the surface area of the vegetative nucleus increased and there was a decrease in the frequency of nuclear pores. The calculated total number of pores remained similar. After activation and pollen-tube growth, the vegetative nucleus retained the same surface area as in the hydrated state but the frequency of nuclear pores decreased; therefore, the calculated total number of pores was significantly lowered. When considered alongside complementary biochemical data, these morphological results indicate that RNA production in the vegetative nucleus decreases following germination.Abbreviations VN vegetative nucleus (nuclei) - GN generativenucleus - GC generative cell - CSLM confocal scanning laser microscope We acknowledge research support by the Biotechnology Action Programm of the Commission of European Communities, and CNR for the fellowship awarded to Dr. Wagner. We would also like to thank Mrs. C. Faleri for the expert technical help.     

Principles and practices of laser scanning confocal microscopy

The laser scanning confocal microscope (LSCM) is an essential tool for many biomedical imaging applications at the level of the light microscope. The basic principles of confocal microscopy and the evolution of the LSCM into today's sophisticated instruments are outlined. The major imaging modes of the LSCM are introduced including single optical sections, multiple wavelength images, three-dimensional reconstructions, and living cell and tissue sequences. Practical aspects of specimen preparation, image collection, and image presentation are included along with a primer on troubleshooting the LSCM for the novice.     

A Fluorescence Staining-Methyl Salicylate Clearing Technique for Demonstrating Pollen Nuclei

Recently several DNA-binding fluotochromes have been used for demonstrating pollennuclei. However, the autofluorescence of pollen wall often obscured the fluorescence of nuclei, thus limited the use of this method. Methyl salicylate (MS) as a clearing agent has shownexcellent effect for observing embryo sac in whole-mounted ovules. This aroused me to trya combination of fluorescent staining with MS clearing in orded to make a better demonstration of the pollen nuclei. Mature 2-celled or 3-celled pollen of several angiosperm species stained with Hoechst 33258(H33258) and cleared (via ethanol dehydration) with MS showed clearcut fluorescence oftheir generative or sperm nuclei and vegetative nucleus. MS greatly decreased the wall fluorescence and increased the transparency of the pollen contents, meanwhile maintained the H33258stained fluorescence, consequently made the nuclei brighter under a darkened background. For example, in sunflower pollen a pair of elongated and winding sperm nuclei whichcould not be identified after simple H33258 staining were quite visible after MS clearing, inartificially germinated pollen tubes, the locomotion of nuclei from pollen grain into the tube,the sequence of generative and vegetative nucle travelling along the tube and the division of generative nucleus into two sperm nuclei could be well followed by this method. The present technique may be adoptable for observations on the processes of microsporogenesis and male gametophyte development, and rogenesis in cultured anthers, and also possiblyfor tracing the nuclear events during pollination-fertilization.     

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

Summary A polyclonal antibody against -1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.Abbreviation TBS Tris-buffered saline - Tris Tris(hydroxy-methyl)-aminomethane - PBS phosphate-buffered saline - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - CLSM confocal laser scanning microscopy - DP degree of polymerization     

Applications of Fluorochjromes to Pollen Biology. Ii. The Dna Probes Ethidium Bromide and Hoechst 33258 in Conjunction with the Callose-Specific Aniline Blue Fluorochrome

Techniques are described for detection of pollen grain and pollen tube nuclei using the fluorescent DNA probes ethidium bromide or Hoechst 33258, in conjunction with the aniline blue fluorochrome sirofluor, which stains the callose component of pollen tube walls and plugs. The DNA probes, which may be used either as vital stains or following fixation, permit discrimination between vegetative and generative or sperm nuclei. Double staining with sirofluor allows location of nuclei within pollen tubes grown in vitro, and when used after pollination enables the viewer to discriminate between nuclei within the pollen tube vs. nuclei of the pistil tissue.