生物显微镜应用技术基础知识(一)

本文首先简述了显微镜的发展历史,以此说明显微镜的发展与各学科发展的关系;文章阐明了显微镜的工作原理、视觉放大率、分辨本领及有效放大率的基本概念,着重指出了数值孔径NA对分辨本领的影响以及关于提高NA从而提高显微镜分辨本领的途径。     

扫描探针显微镜的进展

扫描探针显微镜（SPMs）经过近30年的发展，已经应用到科学研究的各个方面。为适应不同研究的需要，扫描探针显微镜本身的发展也非常迅速。如其中原子力显微镜（AFM）从发明初期的单一的接触工作模式发展到包括可以测量粘弹性的相位模式在内的多种工作模式，同时通用型方面也高度发展，已经形成了一个庞大的高度自动化的扫描探针显微镜的家族。在这个家族中，严格环境控制的扫描探针显微镜的出现，很好的解决了各种务件下对样品的原位观察，环控化扫描探针显微镜的发展已经引起了人们的足够重视，必将成为扫描探针显微镜发展的一个重要方向。新近出现的各种显微镜集成的扫描探针显微镜系列是这个家族中的新成员，这个成员可以同时完成大范围、高分辨和精确定位等各种研究，必将在半导体制造厂的异物检查、金属和绝缘体等表面测定以及生物大分子研究等领域发挥重要作用。扫描探针显微镜的发明和发展促进了一门新兴的高科技——一纳米科学技术的诞生，宣告一个科枝新纪元，纳米科技时代已经来临。     

激光扫描显微镜中的OBIC成像功能

扼要的介绍了激光扫描显微镜在国内外的发展状况,着重介绍了激光扫描显微镜中特殊成像功能OBIC成像的测量方法、基本原理、技术特色和应用前景。     

基于原子晶格为参照标度的纳米计量技术

纳米检测和纳米计量是一项正在发展的科学技术,文中介绍了利用双探头扫描隧道显微镜与原子力显微镜系统,作纳米计量的技术。     

原子力显微镜在细胞生物学中应用的现状

原子力显微镜(AFM)是近十几年来表面成像技术最重要的进展之一.AFM具有原子级分辨率,它的产生和发展为细胞生物学研究提供了一种有效的工具.本文介绍原子力显微镜在细胞生物学中应用的现状,包括细胞固定,细胞成像,力检测及细胞操纵,并对原子力显微镜技术的发展进行展望.     

现代显微成像技术综述

概述了光学宽视场显微镜、共聚焦显微镜、超分辨率显微镜中所应用的现代显微成像技术,对各种传统和先进的显微成像原理进行了总结。光学宽视场显微镜最常用的显微技术有明场成像、暗场成像、相衬成像、偏光成像、微分干涉(DIC)成像、调制对比成像和荧光成像。相衬成像中根据不同的成像结构还有切趾相衬成像。微分干涉除了传统的偏振光照明还有圆偏振光照明(C-DIC)和专用于塑料的微分干涉(PlasDIC)。共聚焦显微镜随着计算机技术和制造技术的发展而有了巨大的发展。除了传统的共聚焦荧光显微镜以外,还有连续反斯托克斯拉曼散射(CARS)共聚焦、多光子共聚焦和白光共聚焦。超分辨率显微镜中主要介绍了受激辐射淬灭(STED)技术和紧随基态淬灭显微技术的单分子返回(GSDIM)技术。     

数码显微镜的设计

介绍了数码显微镜光学系统的设计方法,实现了数码显微镜总体尺寸的缩短,符合数码显微镜小型化的发展方向。采用了多头螺纹调焦机构,改变了传统显微镜由齿轮通过齿条带动镜筒的调焦方式,在调焦过程中具有调焦精细,手感舒适,影象清晰的优点,该结构有利于微型马达的驱动,容易实现数码显微镜调焦的自动化。用精密螺纹连接燕尾槽工作台与调节旋钮,工作台始终在燕尾槽内平稳滑动,克服了数码显微镜在观察移动物体时影像容易晃动的难题。样机研制的结果表明:数码显微镜成象清晰,且结构简单、可靠,光学性能和机械结构均满足设计要求。     

显微镜的市场动向

本文试述显微镜的市场动向,以供发展我国的显微镜生产以及打入国际市场参考。一、中东的显微镜市场:中东盛产石油,近年来由于石油价格的上涨,国民经济收入大为增加,各国正在迅速发展工业并增设了不少学校和医院,对显微镜的需要量逐年增加。下面对沙特阿拉伯和埃及的情况作一介绍。沙特阿拉伯的显微镜进口量是逐年增加的,其中85%以上为政府投标。西德奥普托公司、莱茨/威尔特(Leitz/Wild)公司、美     

扫描探针显微镜在纳米科技中的应用

本文在介绍了扫描探针显微镜的发展和有关纳米科技知识的基础上，论述了扫描显微镜在纳米科技中的应用。     

原子力显微镜发展近况及其应用

扫描隧道显微镜(简称STM)和原子力显微镜(简称AFM),它们也可统称为扫描探针显微镜(简称SPM)。原子力显微镜(AFM) 是近十几年来表面成像技术中最重要的进展之一。与扫描电子显微镜相比,它具有较高的分辨率。本文将讨论原子力显微镜的工作原理、原子力显微镜的发展概况和应用。     

Analytical scanning electron microscopy for solid surface

A scanning electron microscope of ultra-high-vacuum (UHV-SEM) with a field emission gun (FEG) is operated at the primary electron energies of from 100 eV to 3 keV. The instrument can form the images that contain information on surface chemical composition, chemical bonding state (electronic structure), and surface crystal structure in a microscopic resolution of several hundred angstroms (Å) using the techniques of scanning Auger electron microscope, scanning electron energy loss microscope, and scanning low-energy electron diffraction (LEED) microscope. A scanning tunneling microscope (STM) also has been combined with the SEM in order to obtain the atomic resolution for the solid surface. The instrumentation and examples of their applications are presented both for scanning LEED microscopy and STM.     

Optical coherence tomography as a tool for characterization of complex biological surfaces

The advent of scanning electron microscopy has facilitated our understanding of the biology in relation to surface microstructure of many invertebrates. In recent years, interest in biomimetics and bio‐inspired materials has further propelled the search for novel microstructures from natural surfaces. As this search widens in diversity to nurture deeper understanding of form and function, the need often arises to examine rare specimens. Unfortunately, most methods for characterization of the microtopography of natural surfaces are sacrificial, and as such, place limiting constraints on research progress in situations where only a few rare specimens are known, such as the rich resources lodged in natural history museum collections. In this paper, we introduce the use of optical coherence tomography (OCT) as a noninvasive tool for bioimaging surface microtopography of crab shells. The technique enables the capture of microstructures down to micron level using low coherence near‐infrared light source. OCT has allowed surface microtopography imaging on crab shells to be carried out rapidly and in a nondestructive manner, compared to the scanning electron microscope technique. The microtopography of four preserved crab specimens from Acanthodromia margarita, Ranina ranina, Conchoecetes intermedius and Dromia dormia imaged using OCT were similar to images obtained from scanning electron microscope, showing that OCT imaging retains the overall morphological form during the scanning process. By comparing the physical lengths of the spinal structures from images obtained from OCT and scanning electron microscope, the results showed that dimensional integrity of the images captured from OCT was also maintained.     

The molecular machines of DNA repair: scanning force microscopy analysis of their architecture

The application of scanning force microscope (SFM, also called atomic force microscope or AFM) imaging to study the architecture of proteins and their functional assemblies on DNA has provided new and exciting information on the mechanism of vital cellular processes. Rapid progress in molecular biology has resulted in the identification and isolation of proteins and protein complexes that function in specific DNA transactions. These proteins and protein complexes can now be analysed at the single molecule level, whereby the functional assemblies are often described as nanomachines. Understanding how they work requires understanding their structure and functional arrangement in three dimensions. The SFM is uniquely suited to provide three‐dimensional structural information on biomolecules at nanometre resolution. In this review we focus on recent applications of SFM to reveal detailed information on the architecture and mechanism of action of protein machinery involved in safeguarding genome stability through DNA repair processes.     

开放式多功能扫描探针显微镜系统

开放式多功能扫描探针显微镜、集成扫描隧道显微镜、原子力显微镜、横向力显微镜和静电力显微镜．具有接触、半接触和非接触工作模式，可进行作用力、电流、电位、光能量等参数的高度局域综合测量,具有极高的开放性和可扩展性，支持用户进行二次开发。     

Direct examination of chip formation during metal machining by scanning electron microscopy

New insights into the mechanics and morphology of chip formation in metal cutting have been obtained by direct observation of an orthogonal cutting process in progress in a scanning electron microscope. A specimen stage specifically designed for this purpose is described. This stage permits dynamic viewing of all accessible areas of interest at magnifications up to ×5000 during cutting. Permanent records are obtained by videotaping each experiment. A brief summary of results is given, and extensions of the technique of dynamic scanner observation are suggested.     

Developments of scanning probe microscopy with stress/strain fields

An innovative stress/strain fields scanning probe microscopy in ultra high vacuum (UHV) environments is developed for the first time. This system includes scanning tunneling microscope (STM) and noncontact atomic force microscope (NC-AFM). Two piezo-resistive AFM cantilever probes and STM probes used in this system can move freely in XYZ directions. The nonoptical frequency shift detection of the AFM probe makes the system compact enough to be set in the UHV chambers. The samples can be bent by an anvil driven by a step motor to induce stress and strain on their surface. With a direct current (dc) power source, the sample can be observed at room and high temperatures. A long focus microscope and a monitor are used to observe the samples and the operation of STM and AFM. Silicon(111) surface in room temperature and silicon(001) surface in high temperature with stress were investigated to check the performance of the scanning probe microscope.     

近场光学显微镜及其在生物膜方面的应用

本文首先介绍近场光学显微镜的基本原理,然后介绍近场光学显微镜与传统光学显微镜、原子力显微镜、扫描隧道显微镜相比,在生物膜研究方面的优势。并在此基础上着重介绍近场光学显微镜在生物膜方面的应用。     

Quality assessment of atomic force microscopy probes by scanning electron microscopy: correlation of tip structure with rendered images

While image quality from instruments such as electron microscopes, light microscopes, and confocal laser scanning microscopes is mostly influenced by the alignment of optical train components, the atomic force microscope differs in that image quality is highly dependent upon a consumable component, the scanning probe. Although many types of scanning probes are commercially available, specific configurations and styles are generally recommended for specific applications. For instance, in our area of interest, tapping mode imaging of biological constituents in fluid, double ended, oxide-sharpened pyramidal silicon nitride probes are most often employed. These cantilevers contain four differently sized probes; thick- and thin-legged 100 microm long and thick- and thin-legged 200 microm long, with only one probe used per cantilever. In a recent investigation [Taatjes et al. (1997) Cell Biol. Int. 21:715-726], we used the scanning electron microscope to modify the oxide-sharpened pyramidal probe by creating an electron beam deposited tip with a higher aspect ratio than unmodified tips. Placing the probes in the scanning electron microscope for modification prompted us to begin to examine the probes for defects both before and after use with the atomic force microscope. The most frequently encountered defect was a mis-centered probe, or a probe hanging off the end of the cantilever. If we had difficulty imaging with a probe, we would examine the probe in the scanning electron microscope to determine if any defects were present, or if the tip had become contaminated during scanning. Moreover, we observed that electron beam deposited tips were blunted by the act of scanning a hard specimen, such as colloidal gold with the atomic force microscope. We also present a mathematical geometric model for deducing the interaction between an electron beam deposited tip and either a spherical or elliptical specimen. Examination of probes in the scanning electron microscope may assist in interpreting images generated by the atomic force microscope.     

Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering

This paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo‐bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean‐weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed mean‐weight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.     

Real-time image quality assessment with mixed Lagrange time delay estimation autoregressive (MLTDEAR) model

A proposal to assess the quality of scanning electron microscope images using mixed Lagrange time delay estimation technique is presented. With optimal scanning electron microscope scan rate information, online images can be quantified and improved. The online quality assessment technique is embedded onto a scanning electron microscope frame grabber card for real‐time image processing. Different images are captured using scanning electron microscope and a database is built to optimally choose filter parameters. An optimum choice of filter parameters is obtained. With the optimum choice of scan rate, noise can be removed from real‐time scanning electron microscope images without causing any sample contamination or increasing scanning time.