蜈蚣草胚胎发育的观察

用石蜡切片技术观察了蜈蚣草(Pteris vittata L.)的胚胎发育过程.结果表明:蜈蚣草的胚胎由受精卵发育而来.合子最初3次的分裂顺序为X-Y-Z;球形胚最早分化出的组织是基足,其后几乎同时分化出第一叶原始细胞和第一根原始细胞,继而分别分化出第一叶原基和第一根原基,再后来才形成茎干原基;当第一叶、第一根形成时,茎干原基仍不活跃.总结出蜈蚣草的胚胎发育模式图,并讨论了有关分化的作用和意义.     

Recent Developments in Correlative Super-Resolution Fluorescence Microscopy and Electron Microscopy

The recently developed correlative super-resolution fluorescence microscopy (SRM) and electron microscopy (EM) is a hybrid technique that simultaneously obtains the spatial locations of specific molecules with SRM and the context of the cellular ultrastructure by EM. Although the combination of SRM and EM remains challenging owing to the incompatibility of samples prepared for these techniques, the increasing research attention on these methods has led to drastic improvements in their performances and resulted in wide applications. Here, we review the development of correlative SRM and EM (sCLEM) with a focus on the correlation of EM with different SRM techniques. We discuss the limitations of the integration of these two microscopy techniques and how these challenges can be addressed to improve the quality of correlative images. Finally, we address possible future improvements and advances in the continued development and wide application of sCLEM approaches.     

提取蕨类植物蜈蚣草总RNA的一种有效方法

介绍了一种提取蕨类植物蜈蚣草(Pteris vittata L.)总RNA的有效方法.由于蜈蚣草富含多酚和多糖,用普通的RNA提取方法很难获得高质量的RNA.本方法通过优化提取缓冲液的条件来抑制多酚氧化,并利用RNA与多酚、多糖在不同浓度的2-丁氧乙醇中溶解度不同的特性来去除多酚和多糖.本方法简便、快速,所提取的RNA质量较高,可直接用于cDNA合成、cDNA文库的构建以及RACE等分子生物学操作.     

傅氏凤尾蕨配子体发育及其无配子生殖的研究

采用光学显微镜对傅氏凤尾蕨（Pteris fauriei Hieron.）的配子体发育进行了研究。傅氏凤尾蕨孢子深褐色,呈四面体形,三裂缝,极面观为钝三角形,赤道面观近半圆形。孢子自播种3～7 d左右开始萌发,萌发类型为书带蕨型。18 d左右发育为丝状体,大约为6～10个细胞长;25 d左右,丝状体可通过顶端细胞或中间细胞分裂产生片状体。播种40～50 d左右,在片状体的侧面产生分生组织,发育成原叶体,发育类型为水蕨型。雄配子体形状不规则,其上可产生精子器。大型原叶体形状较规则,顶端为心形,发育2～3个月左右,在生长点下方产生无性胚芽,进行无配子生殖。此后不久,在胚芽和生长点之间产生导管连接孢子体和配子体。经多次培养表明傅氏凤尾蕨不产生颈卵器,为专性无配子生殖。     

Fluorescence Microscopy of Fresh Tissue as a Rapid Technique for Assessing Early Injury to Mesophyll

A technique was developed for sectioning fresh red spruce foliage (Picea rubens Sarg.) for use in fluorescence microscopy. This allowed rapid examination of mesophyll in 3-5 mm needle sections. Healthy, ozone treated and cold stressed needles were examined to assess the utility of this technique for early detection of damage. Healthy mesophyll cells fluoresced bright red, while injured cells fluoresced yellow-green in ozone treated needles, and yellow-orange in frozen needles. Shifts in fluorescence wavelengths may be useful for early detection of injury to mesophyll before it is evident by standard light or electron microscopy.     

蜈蚣草是观察减数分裂过程的好材料

以蜈蚣草（Pteris vittata Linn.）为材料,介绍了蜈蚣草孢子母细胞减数分裂的制作方法。该方法操作简单、成功率高;主要特点是在同一孢子囊群中,孢子母细胞的减数分裂不同步,可以同时观察到前期到末期Ⅱ之间各个时期的分裂相。蜈蚣草分布广、取材容易,是观察减数分裂过程的一种好材料。     

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     

蜈蚣草导管分子的观察

对蜈蚣草(Pteris vittata)叶轴中导管分子进行了观察。结果表明,其导管分子十分细长,均具长、且十分倾斜的梯状穿孔板,穿孔没有纹孔膜的残余,与侧壁上的梯纹纹孔有着明显的差异。     

天柱山黄山松种内与种间竞争的研究

研究无菌条件下不同浓度砷对蜈蚣草的孢子萌发、假根和原叶体生长发育的影响。结果表明,培养基质中As浓度大于30mg/L时,孢子萌发速度、假根和原叶体的生长发育受到抑制,假根的着生点位置发生改变;而与高浓度As影响不同的是,低浓度As(30mg/L)可促进孢子变绿的速率和初期原叶体(播后8d内)的生长。因此,高浓度As能抑制配子体生长发育并改变假根着生点位置,而低浓度As则可促进配子体早期的生长。     

三套荧光显微成像系统在光敏剂亚细胞定位研究中的应用比较

目的：对三套荧光显微成像系统在国产新型光敏剂HMME亚细胞定位研究中的应用特点及适用范围进行了比较与评价。方法：分别应用LSCM、CCD、ICCD荧光显微成像系统,选择特异性细胞器荧光探针Rhodamine-123、DIOC6(3)标记细胞内线粒体和内质网。采用细胞器-细胞荧光强度比值法,对HMME进行单细胞内分布的定性与定量研究。结果：LSCM和CCD成像系统能采集到浓度达到160μg／ml时的HMME的荧光图像,获得荧光探针图像信息显示所标记的细胞内线粒体和内质网平均荧光强度比值(J1/J2值)都明显高于细胞内J1/J2值。而ICCD成像系统只需HMME浓度为5μg/ml,荧光图像特点都呈胞浆中荧光强度较高且分布不均,细胞核区荧光较弱的中空现象。ICCD系统对细胞器探针荧光图像在空间分辨上不理想。结论：LSCM与CCD成像系统限于其探测灵敏度,对于弱荧光性光敏剂,适用于其高孵育浓度条件下的亚细胞定位研究。二者获得的结果相一致：孵育24h,HMME在鼠肺内皮细胞线粒体和内质网有分布而几乎不进入细胞核。ICCD成像系统可不受孵育浓度条件的限制,实现光敏剂极微弱荧光的有效探测,但空间分辨率较低。     

蜈蚣草导管分子的观察

对蜈蚣草(Pteris vittata)叶轴中导管分子进行了观察。结果表明,其导管分子十分细长,均具长、且十分倾斜的梯状穿孔板,穿孔没有纹孔膜的残余,与侧壁上的梯纹纹孔有着明显的差异。     

Light Sheet Fluorescence Microscopy: A Review

Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy. LSFMs produce well-registered serial sections that are suitable for three-dimensional reconstruction of tissue structures. Because of a lack of a commercial LSFM microscope, numerous versions of light sheet microscopes have been constructed by different investigators. This review describes development of the technology, reviews existing devices, provides details of one LSFM device, and shows examples of images and three-dimensional reconstructions of tissues that were produced by LSFM.     

Rehabilitation of a tailing dam at Shimen County, Hunan Province: Effectiveness assessment

This study was conducted to assess the effectiveness of phytoremediation on a tailing dam located in Shimen County, Hunan Province. Quadrat survey method was employed to investigate and sample the dominant plant species growing on the rehabilitated tailing dam. The fertilities of the soils were assessed, and concentrations of arsenic and other heavy metals in the plant and soil samples were measured. The results showed that no difference was found on the effect of soil capping with top and non-topsoils for rehabilitation of plants on the tailing dam. After rehabilitation, stable vegetation coverage types were established, 39 plant species were found to grow on the tailing dam, and the minimal area for plant communities was 30 m². The dominant plant species were planted Pteris vittata and natural colonizing Miscanthus sinensis. The contents of organic matter, nitrogen and phosphorus in the soils were low, while the potassium content was at a middle level; however, plots where Legumina plants grew were found to have higher level of nitrogen and phosphorus in the growing soils. Arsenic (As) and Cadmium (Cd) concentrations in the soils were 8 and 7 times of the grade III value of the National Standard for Soil Quality (GB15618-1995), respectively; while in tailings these were 81 and 68 times. The available As concentration in the soils ranged as 3.7–29.5 mg kg^?1, whereas the available As concentration in tailings was as high as 61.1 mg kg^?1. Concentrations for most of the heavy metals were in the normal range of terrestrial higher plants, except As and Cd in P. vittata and M. sinensis, and As in the roots of M. sinensis. It is concluded that phytoremediation project has reduced the ecological and health risks caused by the tailing dam to the ambient environment. However, the plants growing on the tailing dam which contained high As and Cd should be kept from entering into food chain in order to protect the health of local residents.     

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.     

Rehabilitation of a tailing dam at Shimen County, Hunan Province: Effectiveness assessment

This study was conducted to assess the effectiveness of phytoremediation on a tailing dam located in Shimen County, Hunan Province. Quadrat survey method was employed to investigate and sample the dominant plant species growing on the rehabilitated tailing dam. The fertilities of the soils were assessed, and concentrations of arsenic and other heavy metals in the plant and soil samples were measured. The results showed that no difference was found on the effect of soil capping with top and non-topsoils for rehabilitation of plants on the tailing dam. After rehabilitation, stable vegetation coverage types were established, 39 plant species were found to grow on the tailing dam, and the minimal area for plant communities was 30 m². The dominant plant species were planted Pteris vittata and natural colonizing Miscanthus sinensis. The contents of organic matter, nitrogen and phosphorus in the soils were low, while the potassium content was at a middle level; however, plots where Legumina plants grew were found to have higher level of nitrogen and phosphorus in the growing soils. Arsenic (As) and Cadmium (Cd) concentrations in the soils were 8 and 7 times of the grade III value of the National Standard for Soil Quality (GB15618-1995), respectively; while in tailings these were 81 and 68 times. The available As concentration in the soils ranged as 3.7–29.5 mg kg^?1, whereas the available As concentration in tailings was as high as 61.1 mg kg^?1. Concentrations for most of the heavy metals were in the normal range of terrestrial higher plants, except As and Cd in P. vittata and M. sinensis, and As in the roots of M. sinensis. It is concluded that phytoremediation project has reduced the ecological and health risks caused by the tailing dam to the ambient environment. However, the plants growing on the tailing dam which contained high As and Cd should be kept from entering into food chain in order to protect the health of local residents.     

砷超富集植物蜈蚣草原生质体的分离及其抗砷性分析

蜈蚣草（Pteris vittata）是一种砷超富集植物,能够通过根从土壤中吸收砷,并将其输送至羽叶中富集.为了探索蜈蚣草单个细胞在砷积累和砷抗性中的特性,本文首次通过酶解方法获得了这一砷超富集蕨类植物的原生质体,并研究了原生质体在不同浓度砷胁迫下的生活力.结果显示,蜈蚣草原生质体的抗砷性远高于烟草原生质体的抗砷性,与其整体植株的抗性一致.这为探索砷抗性和超富集机理提供了一个新的研究体系.     

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.     

蜈蚣草孢壁发育及其系统学意义

利用光镜、扫描电镜和透射电镜对凤尾蕨科(Pteridaceae)蜈蚣草(Pteris vittata L.)孢壁的形成和发育进行研究。结果表明:蜈蚣草孢子四面体型,极面观钝三角圆形,赤道面观半圆形或超半圆形,近极面具瘤状纹饰和近极脊,远极面具脊并连成网状,具赤道环;孢子具乌毛蕨型外壁,由外壁外层构成纹饰的轮廓;实心型周壁由2层构成,且内层薄、外层具小球体。结合孢子外壁和周壁的发育特征,认为凤尾蕨科与裸子蕨科和水蕨科的亲缘关系较近,支持将裸子蕨科和水蕨科置于凤尾蕨科。     

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.