软X射线显微成像

软X射线显微成像技术为研究生物显微结构开辟了一条新的途径,突破了光学显微镜和电子显微镜在应用上的某些限制。本文评述了软X射线接触显微镜、软X射线成像显微镜、软X射线扫描显微镜以及三维软X射线显微成像和X射线全息术,并介绍了中国科技大学在软X射线接触显微成像方法研究中所取得的成果。     

润滑油中颗粒污染物的数字显微图像测试研究

为检测润滑油被固体机械杂质污染的程度,采用数控载物台和CCD图像捕捉技术,获取颗粒污染物试验样片的数字显微图像,对数字图像进行处理与分割,使计算机对微小颗粒具有可视化和可理解性,使用扫描标号算法测量污染颗粒的尺寸、数量与分布,经数理统计、计算机数据处理,从而确定油液污染度等级,通过数字显微图像技术的应用研究,得到一种新型、快捷、实用、经济的能对润滑油中颗粒污染物进行柔性化定量测试的方法。     

扫描多光谱显微图像的重构及处理

本文介绍了一种新的显微成像一扫描多光谱显微成像的基本原理,着重描述了显微成像中多光谱图像的特点以及图像的获取和重构方法,最后介绍了系统软件的编程。     

基于红蓝双光阑的单物镜立体显微成像系统

为了得到微观动态样品的三维形貌图,提出了基于红蓝双小孔光阑的单物镜立体显微成像方法,并建立了一个结构简单的单物镜立体显微系统。该系统在物镜出瞳处放置分别用红、蓝滤光片覆盖的双小孔光阑,当采用白光照明时,彩色CCD将接收到分别用红光和蓝光表示的两个不同视角的图像。采用数字图像处理方法可获得具有视差的立体像对,基于相位的双目匹配算法可得到样品的三维形貌。实验结果表明,该方法重构了微观样品,其景深达到241.50μm。与现有的双物镜体视显微方法相比,设计的立体显微系统简单且紧凑,利用单次拍摄即可得到微观动态样品的三维形貌图,在显微镜和内窥镜中很有应用前景。     

显微高光谱成像系统的设计

设计出一种基于棱镜 光栅 棱镜组合分光方式的显微高光谱成像实验系统.系统根据推帚式成像光谱仪的原理进行设计,采用棱镜 光栅 棱镜组合元件在后光学系统进行光谱分光,利用高精度载物台自动装置驱动样品进行推扫成像,选用PCI总线作为数据采集的微机接口.整个系统由显微镜、分光计、面阵CCD相机、载物台自动装置以及数据采集与控制模块等几部分组成.系统的光谱范围从400nm到800nm,120个波段,光谱分辨率优于5nm,空间分辨率大约1μm.该系统具有直视性、光谱分辨率高、结构紧凑、成本低等优点;不仅能够提供微小物体在可见光范围的单波段显微图像,而且能够获得图像中任一像素的光谱曲线,实现了光谱技术和显微成像技术的结合,成功的将成像光谱技术应用到显微领域,可广泛应用于临床医学、生物学、材料学、微电子学等学科领域.     

显微荧光光谱成像仪的研究与设计

将光谱分析技术快速、灵敏、准确等独特的优点,与光学成像技术的定位记录特点融合、构成光谱成像技术,可以提供分析试样中各种化学或生化成份的分布图像,因而可以获得定性、定量和定位综合、更丰富的分析信息。报导在商售落射荧光显微镜基础上,实现显微镜荧光光谱成像技术的理论基础、系统组成、设计技术关键等一系列研究工作,设计研制成了可在250~680nm波长范围内自动扫描的光纤激发显微荧光光谱成像仪样机,并获得了满意的实验结果。     

浅谈共聚焦显微技术

共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用。常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高。     

生物大分子超微结构研究中的原子力显微术

原子力显微镜作为第三代显微探测工具,具有原子级的空间分辨率,其样品制备方法简单易行,可在离体的近生理条件下直接观测生物样品及其动态变化过程,能够对样品进行力学操纵,在观察生物大分子的结构和生物力学特性上具有显著的优势。本文尝试从蛋白质、核酸、多糖的超微结构和力学特性的研究角度入手,期望向读者展现出原子力显微镜在大分子生物学研究中的应用前景。     

基于小波分析的显微序列图像合成系统

由于显微镜物镜焦深范围有限,故造成显微图像局部模糊,研究人员对图像的理解不完整等问题。根据聚焦评价函数分布,采用小波分析,将一序列局部聚焦图像合成全焦图像。改进的小波融合算法得到的融合图像极大地保留了系列图像聚焦部分的信息,合成全焦图像。由此得出的聚焦合成图像使研究人员可直接测量所需的显微结果。     

近红外显微成像技术

以烟草混合样品为例简要介绍近红外显微成像技术和近红外显微镜的使用,介绍与之相关的实现化学成分鉴别的图像处理方法及图像显示方式。     

Scanning luminescence X-ray microscopy: Imaging fluorescence dyes at suboptical resolution

Scanning luminescence X-ray microscopy is based on the use of the very small focused probe of a scanning X-ray microscope to stimulate visible light emission from phosphors and dyes. Using an undulator X-ray source and a Fresnel zone plate to produce a focused X-ray probe, images of P31 phosphor grains with a resolution of 50–75 nm have been obtained, and luminescence from polystyrene spheres loaded with 50–100 μmol/g of fluorescent dye has been imaged. The resolution was not limited by the focused X-ray probe (the microscope has imaged features at 36-nm spacing in transmission mode) but by dark noise and the low net efficiency of the luminescence detection system used for this investigation. This technique may make it possible to image dye-tagged sites of biochemical activity at the resolution of the X-ray microscope in wet, unsectioned, and unfixed cells, especially with soft X-ray optimized dyes. Because the image is formed from the detection of signal against a dark background, calculations suggest that the radiation dose for luminescence imaging of dye-tagged features should be 2–22 times lower than it is in transmission X-ray microscopy. A possible extension of the technique for three-dimensional imaging at the transverse resolution of the X-ray microscope is described, where visible light collection optics might be used to obtain submicrometre axial resolution.     

Coaxial arrangement of a scanning probe and an X-ray microscope as a novel tool for nanoscience

We report on the design and first tests of a novel instrument aimed at combining the benefits of scanning force microscopy with those of X-ray spectroscopy. For this we built an instrument combining a scanning transmission X-ray microscope with a beam-deflection atomic force microscope in a coaxial geometry. This allows to combine X-ray absorption spectroscopy and high resolution topography in-situ. When replacing the conventional scanning probe tip by a coaxially shielded tip the instrument will allow detection of the photoelectrons produced by resonant X-ray absorption. This could yield spectroscopic information with a spatial resolution approaching the values achievable with atomic force microscopy.     

Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography

Using a cryo scanning transmission X-ray microscope ( Maser, et al . (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc . 197, 68–79), we have obtained tomographic data-sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three-dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.     

Soft X-ray spectroscopy from image sequences with sub-100 nm spatial resolution

A method is described whereby a sequence of X-ray images at closely spaced photon energies is acquired using a scanning transmission X-ray microscope, and aligned. Near-edge absorption spectra can then be obtained both from large, irregular regions, and from regions as small as the spatial resolution of the microscope (about 40 nm in the examples shown here). The use of the technique is illustrated in examination of a layered polymer film, a micrometeorite section, and an interplanetary dust particle section.     

The spatial resolution of imaging using core-loss spectroscopy in the scanning transmission electron microscope

The 'delocalization' of inelastic scattering is an important issue for the ultimate spatial resolution of core-loss spectroscopy in the electron microscope. This paper investigates the resolution of scanning transmission electron microscopy images for single, isolated atoms. Images are simulated from first principles using a nonlocal model for electron core-loss spectroscopy. The role of the width of the probe relative to the delocalization of the underlying ionization interaction is considered.     

Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: I. Instrumentation, imaging and spectroscopy

We have developed a cryo scanning transmission X-ray microscope which uses soft X-rays from the National Synchrotron Light Source. The system is capable of imaging frozen hydrated specimens with a thickness of up to 10 μm at temperatures of around 100 K. We show images and spectra from frozen hydrated eukaryotic cells, and a demonstration that biological specimens do not suffer mass loss or morphological changes at radiation doses up to about 10¹⁰ Gray. This makes possible studies where multiple images of the same specimen area are needed, such as tomography ( Wang et al. (2000 ) Soft X-ray microscopy with a cryo scanning transmission X-ray microscope: II. Tomography. J. Microsc . 197, 80–93) or spectroscopic analysis.     

Software image alignment for X-ray microtomography with submicrometre resolution using a SEM-based X-ray microscope

Improved X-ray sources and optics now enable X-ray imaging resolution down to ∼50 nm for laboratory-based X-ray microscopy systems. This offers the potential for submicrometre resolution in tomography; however, achieving this resolution presents challenges due to system stability. We describe the use of software methods to enable submicrometre resolution of approximately 560 nm. This is a very high resolution for a modest laboratory-based point-projection X-ray tomography system. The hardware is based on a scanning electron microscope, and benefits from inline X-ray phase contrast to improve visibility of fine features. Improving the resolution achievable with the system enables it to be used to address a greater range of samples.     

Optimization of scanning transmission X-ray microscopy for the identification and quantitation of reinforcing particles in polyurethanes

The morphology, size distributions, spatial distributions, and quantitative chemical compositions of co-polymer polyol-reinforcing particles in a polyurethane have been investigated with scanning transmission X-ray microscopy (STXM). A detailed discussion of microscope operating procedures is presented and ways to avoid potential artifacts are discussed. Images at selected photon energies in the C 1s, N 1s and O 1s regions allow unambiguous identification of styrene-acrylonitrile-based (SAN) copolymer and polyisocyanate polyaddition product-based (PIPA) reinforcing particles down to particle sizes at the limit of the spatial resolution (50 nm). Quantitative analysis of the chemical composition of individual reinforcing particles is achieved by fitting C 1s spectra to linear combinations of reference spectra. Regression analyses of sequences of images recorded through the chemically sensitive ranges of the C 1s, N 1s and O 1s spectra are used to generate quantitative compositional maps, which provide a fast and effective means of investigating compositional distributions over a large number of reinforcing particles. The size distribution of all particles determined by STXM is shown to be similar to that determined by TEM. The size distributions of each type of reinforcing particle, which differ considerably, were obtained by analysis of STXM images at chemically selective energies.     

Ultra-high resolution with off-axis STEM holography

It has previously been demonstrated that the scheme of Rodenburg and colleagues for deriving ultra-high resolution images from arrays of nanodiffraction patterns recorded in a scanning transmission electron microscopy instrument is capable of producing reconstructed images with an improvement in resolution by a factor greater than two, but the method is limited to weak-phase objects and the desired image is accompanied by an unwanted background. It is now shown that these limitations of the method can be avoided if the technique is combined with an off-axis electron holography scheme.     

NanoSIMS imaging of Bacillus spores sectioned by focused ion beam

Preparation and sectioning of bacterial spores by focused ion beam and subsequent high resolution secondary ion mass spectrometry analytical imaging is demonstrated. Scanning transmission electron microscopy mode imaging in a scanning electron microscope is used to show that the internal structure of the bacterial spore can be preserved during focused ion beam sectioning and can be imaged without contrast staining. Ion images of the sections show that the internal elemental distributions of the sectioned spores are preserved. A rapid focused ion beam top‐sectioning method is demonstrated to yield comparable ion images without the need for sample trenching and section lift‐out. The lift‐out and thinning method enable correlated transmission electron microscopy and high resolution secondary ion mass spectrometry analyses. The top‐cutting method is preferable if only secondary ion mass spectrometry analyses are performed because this method is faster and yields more sample material for analysis; depth of useful sample material is ～300 nm for top‐cut sections versus ～100 nm for electron‐transparent sections.