Applications of Environmental Scanning Electron Microscopy (ESEM) in botanical research

Abstract

Conventional Scanning Electron Microscopy (SEM) is limited by artefacts from sample preparation, while Environmental Scanning Electron Microscopy (ESEM) permits observations of hydrated, non-conductive samples without any preparation. In this short review, the two systems are described and some examples given. In addition, a study of trace element localization by X-ray ESEM microanalysis in Azolla caroliniana cultured in the presence of trace elements is presented. The highest concentration occurred in roots and stem. Leaves showed lower accumulation, with concentrations decreasing from the base to the apex of the shoot, and sharp differences between ventral and dorsal lobes of single leaves, the former accumulating more than the latter. The epidermal cells in the ventral lobes of basal leaves were largely lost in treated plants. The differential localisation of trace elements in the plant protected the dorsal lobes, which are the main photosynthetic part of the plant, the nitrogen-fixing cyanobacterial colonies and the apical meristems from potentially adverse effects of trace elements.     

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。     

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using “off-the-shelf” microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.     

激光扫描共聚焦显微镜系统及其在细胞生物学中的应用

激光扫描共聚焦显微镜是近十年发展起来的医学图象分析仪器,现已广泛应用于荧光定量测量、共焦图象分析、三维图象重建、活细胞动力学参数监测和胞间通讯研究等方面。其性能为普通光学显微镜质的飞跃,是电子显微镜的一个补充。本文以美国Ｍｅｒｉｄｉａｎ公司的ＡＣＡＳＵＬＴＩＭＡ３１２为例简要介绍了激光扫描共聚焦显微镜系统的结构、功能和生物学应用前景。     

用近场光学显微镜观察红细胞的自发荧光

传统的自体荧光检测技术均是对大量细胞或组织进行检测,而近场光学显微技术由于具有较高分辨率和能够同时获取样品的外部形貌和光学信息等特点,有望成为一种研究单个细胞自体发光机理、疾病诊断和检测单个细胞自体荧光光谱的新技术。本文通过应用近场光学显微镜观察不同形状红细胞的外部形貌和光学信息,来初步探讨近场光学显微技术在这方面的应用前景。     

Near-Field Scanning Optical Microscopy, a Siren Call to Biology

Near-field illumination of a sample with visible light can resolve features well beyond the resolution of conventional, far-field microscopes. Near-field scanning optical microscopy (NSOM) then has the potential of extending the resolution of techniques such as fluorescent labeling, yielding images of cell structures and molecules on the nanoscale. However, major problems remain to be solved before NSOM can be easily used for wet biological samples. The most significant of these is control of the distance between near-field aperture and the sample surface. Hence, while NSOM promises much, its application to biology is about where electron microscopy was 40 or 50 years ago.     

食蚜瘿蚊触角的扫描电镜观察

用扫描电子显微镜对食蚜瘿蚊角角进行了观察.结果显示,雌雄触角都为14节,其中雄性约为2000μm,雌性约为1 050μm.电镜下可观察到食蚜瘿蚊触角有6种类型的感受器,即：刺形感受器、毛形感受器、锥形感受器、腔形感受器、柱形感受器和环丝.刺形感受器较长,约67.5μm,基部有膜状的窝.毛形感受器长约61μm,末端弯曲.锥形感受器呈钉状着生在表皮上,长约4.7μm.腔形感受器呈凹陷状,腔的直径约为1.2μm.柱形感受器着生在雄虫鞭节的第二亚节,长约21μm,直径约为1.5μm.环丝,是瘿蚊类昆虫触角中特殊的结构,它通过着生在一系列腔中的的短梗,连结成环状附着在触角各亚节的表面.刺形和锥形感受器在数量上,雌雄之间差别不大;柱形感受器只在雄虫中发现;雄虫触角上的腔形感受器在数量上要比雌虫多.     

Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy

Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.     

激光扫描共聚焦显微镜荧光探针的选择和应用

激光扫描共聚焦显微镜是检测生物荧光信号的最新技术手段。不仅广泛用于荧光定性、定量测量,还可用于活细胞动态荧光监测、组织细胞断层扫描、三维图象重建、共聚焦图象分析、荧光光漂白恢复、激光显微切割手术等。本文拟就激光扫描共聚焦显微镜常用的检测内容及其相关荧光探针的选择和应用做一简单的介绍。     

Imaging of Oil/Monoglyceride Networks by Polarizing Near-Field Scanning Optical Microscopy

Polarizing near-field scanning optical microscopy (NSOM) was applied for visualization of lipid coagel structures. The technique ensures obtaining polarization contrast images at micro- and nanoscale resolution. Comparison to the polarizing light microscopy images revealed that the same fractal structural organization persists also at submicron scale, at the level of primary ordered structures creation. Many long birefringent needle-shaped primary crystallites were imaged in the corn oil:monoglyceride samples, and lower amount of smaller oval-shaped primary crystallites—in the olive oil:monoglyceride samples. Unlike atomic force microscopy, polarizing NSOM brought direct evidence on the physical state of specific features. Compared to the polarizing light microscopy, polarizing NSOM provided additional information on the structural organization of oil–monoglyceride coagels at the micro- and submicron scale.     

Sensing Nanometer Depth of Focused Optical Fields with Scanning Surface Plasmon Microscopy

Evanescent wave imaging has been developed in the past decades for discriminating sub-micronic structures confined on a planar surface from bulk medium. However, this imaging method assumes a correct orientation of the objective lens versus the normal of the sample plane (null tilt angle) for a uniform evanescent illumination of the sample. Here, we propose to use the $V(Z)$ response of a heterodyne scanning microscope coupled to a high numerical aperture lens to compensate the tilt, scanning images in three dimensions. This method can be used in standard glass to dielectric reflection regime or coupled to surface plasmon resonance. We show that in the second case, we can afford tilt angle corrections better than 10 $^{-4}$ radian. As an illustration, this method is applied to a patterned surface with nanometer-squared silica islets and to adsorbed nanoparticles.     

Scanning Electron Microscopy (SEM) of the Chick Embryo Primitive Streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2–5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the primitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arranged cells with long, thin processes.
Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.     

A Scanning Electron Microscopy Study of the Seed and Post-seminal Development in Angelonia salicariifolia Bonpl. (Scrophulariaceae)

The external morphology of seeds and post-germination developmentalstages of Angelonia salicariifolia Bonpl. (Scrophulariaceae)were investigated using scanning electron microscopy. Some structuralfeatures of the seed exotesta and seedling in Angelonia arepresented for the first time and are of potential taxonomicvalue for this neotropical genus. The seeds are very small (0.9–1.7mm long and 0.5–0.9 mm wide), ovate, with a reticulate-crestedexotesta, reticules arranged uniformly in longitudinal rows,with a high density of microcilia-like projections on the cellwall of the reticule base and on the edge of the crests. Thehilum is located beside the micropyle at the narrow end of theseed. Germination is epigeal. During germination the radicledevelops, followed by elongation of the hypocotyl and primaryroot. At this stage dense root hairs develop on the lower partof the hypocotyl. The apical bud—located between the cotyledons—beginsto develop after the cotyledons have unfolded. The cotyledonsare equal in size, sessile and ovate. The seedlings have twotypes of trichomes, one characteristic of the cotyledons andfirst pair of leaves (glandular, sessile, four-celled head withquadrangular shape) and the other characteristic of the hypocotyland epicotyl (stalked, erect, elongate and three-celled withdome-shaped unicellular head). Copyright 2001 Annals of BotanyCompany Seed morphology, exotesta-ornamentation, micromorphology, post-seminal stages, seedling, trichomes, SEM, Angelonia salicariifolia Bonpl., Scrophulariaceae     

A Simple Drying Method for Field Emission Scanning Electron Microscopy for Chromosomes

Hexamethyldisilazane treatment and subsequent air drying of spread plant chromosomes is compared with critical point drying. The two procedures are equivalent for preparing chromosomes for examination by field emission scanning electron microscopy at low voltage.     

同时应用近场光学显微镜的透射和反射模式研究单个细胞的光学性质

通常认为．在近场光学显微技术的光收集模式中,观察透光性好的样品时采用透射模式．研究不透明样品时采用反射模式。本文同时采用透射和反射两种模式观察透明性较好的PCI2细胞和淋巴细胞样品．初步研究单个细胞的反射、吸收、透射和荧光等光学性质,以促进组织光学和激光生物医学等领域的进一步发展。细胞光学的时代就要到来。     

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets¹, monitor dynamics in living cells^2-4, and visualize the three dimensional evolution of entire organisms^5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo⁷ and are currently being applied to disease imaging and diagnosis in clinical settings^8,9.Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole¹, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.Download video file.^{(90M, mov)}     

基于扫描电镜技术的天葵属(毛茛科)花器官发生研究

毛茛科天葵属为东亚特有类群,但其花器官的发生过程仍不清晰。该研究利用扫描电子显微镜观察了天葵［S. adoxoides (DC.) Makino］花器官的发生过程,以揭示毛茛科花形态的多样性和演化规律,为进一步探讨天葵属与近缘类群的亲缘关系提供发育形态学证据。结果表明：(1)天葵萼片、花瓣和雄蕊均为螺旋状发生,轮状排列;不育雄蕊的数目和位置不定,心皮轮状发生。(2)天葵萼片原基为宽阔的新月形,其他花器官为窄的半球形。(3)天葵花发育后期,花瓣有延迟发育现象,花瓣原基基部发育为浅囊状,心皮原基马蹄形对折,胚珠倒生、双珠被、具胎座附属物。(4)天葵属与耧斗菜属、尾囊草属的花发育性状存在相似性,支持分子系统学证据的三者近缘的观点;天葵属的花性状的特殊表现为：花直径较小,雄蕊、不育雄蕊和心皮数目较少,花器官没有形成明显的直列线,内珠被较长等。     

佩妃延腹榕小蜂雌蜂触角感器的扫描电镜观察

佩妃延腹榕小蜂是一种与鸡嗉子传粉榕小蜂同步寄生于接收期榕果的非传粉专性寄生蜂。为探索佩妃延腹榕小蜂寄主定位机制,应用扫描电镜观察了其雌蜂触角感器的类型、分布和超微形态。结果显示:佩妃延腹榕小蜂雌蜂触角呈膝状,长775.50μm±38.39μm,由柄节、梗节和11个鞭小节组成的鞭节构成。触角上共发现5种感器类型,分别为毛形感器、刺形感器(Ⅰ型和Ⅱ型)、多孔板形感器、锥形感器、栓锥型乳突状感器。其中,刺形感器Ⅰ和多孔板形感器是其主要感器,前者集中分布于鞭节的第4~第8鞭小节,后者分布于第4~第11鞭小节。结合触角感器的形态、分布和佩妃延腹榕小蜂的产卵行为,对各触角感器的功能进行了探讨。