Feulgen Staining of Intact Plant Tissues for Confocal Microscopy

A method was developed to prepare plant structures for confocal laser scanning microscopy by combining Feulgen staining with pararosaniline and embedding in LR White_TM. This procedure preserves intact, delicate structures for three-dimensional imaging without loss from sectioning or squashing, and the slides can be viewed several times without serious photobleaching.     

用激光共聚焦显微术检测哺乳动物卵母细胞中纺锤体相关蛋白的方法(英)

哺乳动物卵母细胞是发育生物学和细胞生物学的重要研究对象.激光共聚焦显微术是检测卵母细胞中纺锤体相关蛋白的有用技术,但是针对哺乳动物卵母细胞的特点需要做许多调整以得到理想结果.其中,卵母细胞的准备、固定、透膜、脂类抽提、抗体孵育步骤十分关键.另外,不同物种的卵母细胞也有差异,在进行激光共聚焦研究时需要做出相应的调整.     

用激光共聚焦显微术在小鼠卵母细胞中检测蛋白激酶C

用免疫荧光化学与激光共聚焦显微术结合的方法研究了小鼠卵母细胞中蛋白激酶C(PKC)α和βⅠ的表达和定位,以及蛋白激酶C(PKC)和皮质颗粒的双标记,探讨了以哺乳动物卵母细胞为实验对象进行免疫荧光共聚焦显微研究的简便方法.结果发现,PKC α和βⅠ在小鼠生发泡期和MⅡ期卵母细胞中都有表达,但表达部位存在差异.说明采用改进的激光共聚焦显微术,可以方便、灵敏地检测特异蛋白质在卵母细胞中的表达部位,从而为生殖、发育研究提供有效手段.     

An Improved Mounting Method for Observation of Thick Specimens Using Confocal Microscopy

When applying aqueous media to mount thick specimens, air bubbles occur frequently between coverslips and slide glasses. This results in an increase of background signal by confocal microscopy. Using Spurr's resin as a mounting medium, we could observe thick specimens with oil immersion objective lens without the use of coverslips, then avoid air bubbles near the specimen. Fading of the fluorescence appeared to be less in specimens mounted by the resin than in specimens mounted by aqueous media. Further, the new method increased depth of field so that more planes of specimens could be analyzed.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

共聚焦激光扫描显微镜在发育生物学中的应用

共聚焦激光扫描显微镜以高空间分辨率、非介入无损伤性连续光学切片、实时动态观察等优越性,应用于生物医学众多领域中。本文主要论述共聚焦激光扫描显微镜在发育生物学中的应用。     

活细胞钙动态的共聚焦扫描显微镜检测技术

共聚焦激光扫描显微镜（Confocal Laser Scarming Microscope,CLSM）广泛应用于活细胞内钙敏感探针标记的钙水平的动态测量。较之传统的显微镜CLSM在钙成像分析上有着不可比拟的优越性,但也存在一些缺陷,近些年陆续出现了一些针对这些缺陷的改善措施,如比率法、葡聚糖探针及其他一些新技术与共聚焦显微镜的联合应用等,并且出现了诸如双光子显微镜等新型激光共聚焦显微镜。随着共聚焦钙成像技术的不断发展进步,其今后的应用前景将会越越广阔。     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Stokes参量共焦显微术用于弱各向异性物质的成像研究

提出将基于Stokes参量的偏振共焦显微成像技术应用于弱各向异性物质的成像研究。通过将分振幅Stokes参量测量法与共焦扫描成像技术相结合的方法,得到了基于Stokes参量测量的偏振共焦显微成像系统。利用该系统对具有弱各向异性的生物组织样品进行逐点测量,通过四个通道同时测量获得全部的Stokes参量。再以计算得到的偏振参量作为成像物理量进行图像重建,获得对应Stokes参量、偏振度、相位差、方位角和椭率角的空间分布图像,从而对生物组织实现细胞水平的偏振显微成像研究。实验结果表明:基于Stokes参量的偏振共焦显微成像技术能够获得弱各向异性的生物组织样品的显微图像,并通过比较样品的Stokes参量及相关偏振参量的分布图像,提取样品全部的偏振信息,从而为生物组织的特性研究提供更丰富的信息。     

中国对虾卵子活化及卵裂的激光扫描共聚焦显微镜研究(英文)

有报导,与其它动物不同,对虾的卵子一接触海水、不经过受精,就可以被活化。但是,只有受精卵才能卵裂发育。而且,各种对虾卵子内皮质棒及皮层颗粒的形态结构、活化过程各有不同。本文就中国对虾卵子的活化及卵裂过程进行了激光扫描共聚焦显微镜（ＬＳＣＭ）研究。该技术可以清晰地显示细胞的立体图像。采集中国对虾,人工诱导产卵,未受精卵取自产卵虾体内,受精卵分期分批取样,固定。供试样品经ＰＢＳ漂洗,ＡＯ、ＦＩＴＣ和ＰＩ染色,ＬＳＣＭ观察、图像处理。刚产出的、未活化卵子形态不规则,皮层内富含皮质棒和皮层颗粒（Ｆｉｇ１）。不论受精与否,产入海水的卵子都能被激活并恢复减数分裂。活化后的卵子释放出皮层囊泡内的皮质棒（Ｆｉｇｓ２＆３）。释放出的皮质棒转变为均质的胶质层（Ｆｉｇｓ４＆５）。随后,皮层颗粒释放并在卵子表面转化为均质化的孵化膜。卵子开始举起,与孵化膜之间形成卵周隙。卵周隙内具有絮状物质（Ｆｉｇｓ６,７＆８）。卵子产出后笫８～１０ｍｉｎ,经过减数分裂,第一极体排放,并随着孵化膜的举起而被推向卵外（Ｆｉｇ５）。笫２０ｍｉｎ,第二极体排放,并由卵子表面到达孵化膜的内缘（Ｆｉｇ６）。在２０℃水温条件下,受精卵产入海水中６０     

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。     

Confocal microscopy of whole ovules for analysis of reproductive development: the elongate1 mutant affects meiosis II

Analysis of female meiosis (megasporogenesis) and embryo sac development (megagametogenesis) in angiosperms is technically challenging because the cells are enclosed within the nucellus and ovule tissues of the female flower. This is in contrast to male sporogenesis and gametogenesis where development can readily be observed through the easily dissectable developing anthers. Observation of embryo sac development is a particular problem in crassinucellate ovules such as those of maize. To overcome the problems in observing reproductive development, we developed a simple Feulgen staining procedure optimized for use with confocal microscopy to observe reproductive progression in the crassinucellate ovules of maize. The procedure greatly facilitates the observation of nuclei and cell structures of all stages of megasporogenesis and embryo sac development. The high resolution obtained using the technique enabled us to readily visualize chromosomes from individual cells within ovule tissue samples of maize. A propidium iodide staining technique was also used and compared with the Feulgen-based technique. Static cytometry of relative DNA content of individual nuclei was possible using Imaris software on both Feulgen and propidium iodide-stained samples. The techniques also proved successful for the observation of Arabidopsis and Hieracium aurantiacum female gametophyte and seed development, demonstrating the general applicability of the techniques. Using both staining methods, we analysed the maize meiotic mutant elongate1, which produces functional diploid instead of haploid embryo sacs. The precise defect in meiosis from which diploid embryo sacs arise in elongate1 has not previously been reported. We used confocal microscopy followed by static cytometry using Imaris software to show that the defect by which diploid embryo sacs arise in the maize mutant elongate1 is the absence of meiosis II with one of the dyad cells directly initiating megagametogenesis.     

3D Confocal Laser Scanning Microscopy for the Analysis of Chlorophyll Fluorescence Parameters of Chloroplasts in Intact Leaf Tissues

We analyzed the chlorophyll fluorescence parameters in a 3Dcellular arrangement in vivo by using a modified Nipkow disk-typeconfocal laser scanning microscope (CLSM). We first definedthe 3D values of _PSII (photochemical yield of PSII) and NPQ(non-photochemical quenching) in mesophyll, epidermal and guardcell chloroplasts from the leaf surface to several tens of micronsin depth. We also used this CLSM method to analyze the relationshipsbetween actinic light intensity and the chlorophyll fluorescenceparameters for Boston fern and broad bean leaf specimens. Asthe actinic light intensity increased, the mean _PSII valuesdecreased and the NPQ values increased in all chloroplasts ofBoston fern and broad bean leaf. These values differed withcell type and species. The Boston fern chloroplasts had lower_PSII values than the broad bean chloroplasts, and vice versafor the NPQ values. The _PSII values of Boston fern chloroplastsdecreased in the order mesophyll, epidermal and guard cell chloroplasts.The NPQ values decreased in the order guard cell, mesophylland epidermal chloroplasts, except at 12 µmol m^–2s^–1 actinic light, when the mesophyll value was slightlylower than that of the epidermis. The trend in the _PSII andNPQ values of broad bean mesophyll and guard cell chloroplastswas opposite to that of Boston fern chloroplasts. As 3D CLSMcan provide the _PSII and NPQ values of each chloroplast in a3D cellular arrangement, this method has potential for investigatingdifferences in the functions of chloroplasts in vivo.     

Semiquantitative Confocal Laser Scanning Microscopy Applied to Marine Invertebrate Ecotoxicology

Confocal laser scanning microscopy (CLSM) represents a powerful, but largely unexplored ecotoxicologic tool for rapidly assessing in vivo effects of toxicants on marine invertebrate embryo quality and development. We describe here a new semiquantitative CLSM approach for assessing relative yolk quantity in marine invertebrate embryos (harpacticoid copepods) produced by parents reared from hatching to adult in the polycylic aromatic hydrocarbon chrysene. This method is based on fluorogenic labeling of embryo yolk and subsequent statistical analysis of areal pixel intensities over multiple Z-series using a general linear model (GLM)–nested analysis of variance. The fluorescent yolk-labeling method described here was able to detect statistically significant differences in yolk concentrations in marine copepod (Amphiascus tenuiremis) eggs or embryos from females exposed to ultraviolet light and chrysene-contaminated sediments. Yolk intensities in embryos from females cultured throughout their life cycles in clean sediments were statistically identical with or without UV exposure. In constrast, yolk intensities in embryos of females cultured throughout their life cycle in chrysene-contaminated sediments were significantly higher in the non-UV-exposed treatment with chrysene at 2500 ng/g sediment (65.7% higher) and the UV-exposed treatment with chrysene at 500 ng/g sediment (76.6% higher).     

利用转盘共聚焦显微镜进行快速实验的新方法

转盘共聚焦显微镜是快速激光共聚焦显微镜的一种,与传统的激光共聚焦显微镜相比具有一些相同点,也有其特有的优势。本文主要介绍转盘共聚焦显微镜的基本原理及如何利用转盘共聚焦显微镜进行快速实验及应用实例,并与传统激光共聚焦显微镜进行比较。转盘共聚焦显微镜具有速度快、灵敏度高、对样品光损伤和光淬灭程度低、操作灵活简单,是随着实验技术发展使用越来越广泛的实验仪器。     

Bacterial Maceration for Feulgen Squash Preparations of Plant Tissues

An active pectinolytic extract obtained from Bacterium aroideae is an effective macerating medium for making Feulgen squash preparations from materials which are not sufficiently macerated by Feulgen hydrolysis in normal hydrochloric acid. To obtain the extract, the bacterium is cultured in a suitable liquid medium for 4 days at about 25°C. The cultures are then bulked, toluene is added and the whole is shaken and left overnight. The liquid may then be cleared by centrifuging and stored ready for use in small volumes over dry ice. The tissue to be macerated is fixed, bleached if necessary, washed and soaked in the active extract 2-4 days before Feulgen staining. The organism is readily obtainable, is easy to culture and the strain remains constant. This, coupled with the fact that the extract, once prepared, retains its full activity for at least 6 mo makes it a convenient source of pectinolytic enzyme for the plant cytologist.     

ConfocalVR: Immersive Visualization for Confocal Microscopy

ConfocalVR is a virtual reality (VR) application created to improve the ability of researchers to study the complexity of cell architecture. Confocal microscopes take pictures of fluorescently labeled proteins or molecules at different focal planes to create a stack of two-dimensional images throughout the specimen. Current software applications reconstruct the three-dimensional (3D) image and render it as a two-dimensional projection onto a computer screen where users need to rotate the image to expose the full 3D structure. This process is mentally taxing, breaks down if you stop the rotation, and does not take advantage of the eye's full field of view. ConfocalVR exploits consumer-grade VR systems to fully immerse the user in the 3D cellular image. In this virtual environment, the user can (1) adjust image viewing parameters without leaving the virtual space, (2) reach out and grab the image to quickly rotate and scale the image to focus on key features, and (3) interact with other users in a shared virtual space enabling real-time collaborative exploration and discussion. We found that immersive VR technology allows the user to rapidly understand cellular architecture and protein or molecule distribution. We note that it is impossible to understand the value of immersive visualization without experiencing it first hand, so we encourage readers to get access to a VR system, download this software, and evaluate it for yourself. The ConfocalVR software is available for download at http://www.confocalvr.com, and is free for nonprofits.     

Confocal microscopy of hair

Confocal microscopy is an excellent method for studying the localization of fluorescent stains. Used in this way, superior 3D images can be obtained from multiple optical sections with very shallow depth of field. The main advantage of this technique is that the sample is not damaged. We have taken serial confocal sections of hair and via specific image enhancement routines have obtained high-quality 3D images enabling the visualization of cuticle scale and its pattern of distribution. This has been done on various types of hair: bleached, permed and in certain pathological conditions. This first step will allow us to characterize the hair surface in terms of its roughness, and the distribution and form of cuticular scale, parameters that have potential in the assessment of dermocosmetic efficacy.     

双光子及共聚焦激光扫描显微术研究天花粉蛋白对人绒癌细胞的作用机制

天花粉蛋白(trichosanthin, TCS)是从中草药栝楼根中提取的一种核糖体失活蛋白,具有抗肿瘤和抗HIV功能.应用双光子及共聚焦激光扫描显微术结合特异性荧光探针Hoechst 33342、2′,7′-二氯荧光黄双乙酸酯 (DCFH-DA)、Indo-1和Fluo 3-AM,首次同时观察了TCS诱导人绒癌细胞（JAR细胞）凋亡过程中活性氧自由基（ROS）和细胞内钙离子浓度（［Ca²⁺］_i）的变化,实验结果表明TCS引起的［Ca²⁺］_i升高和ROS形成参与了TCS诱导的JAR细胞凋亡,并且ROS形成和［Ca²⁺］_i升高有关.共聚焦激光扫描显微术的研究结果表明,［Ca²⁺］_i升高不是导致ROS形成的主要原因,TCS诱导产生的ROS可能是通过TCS与JAR细胞膜表面受体作用介导的.