Visualizing soft tissue in the mammalian cochlea with coherent hard X-rays

This paper concerns an important aspect of current developments in medical and biological imaging: the possibility for imaging soft tissue at relatively high resolution in the micrometer range or better, without tedious and/or entirely destructive sample preparation. Structures with low absorption contrast have been visualized using in-line phase contrast imaging. The experiments have been performed at the Advanced Photon Source, a third generation source of synchrotron radiation. The source provides highly coherent X-ray radiation with high photon flux (>10(14) photons/s) at high photon energies (5-70 keV). Thick gerbil cochlear slices have been imaged and were compared with those obtained by light microscopy. Furthermore, intact gerbil cochleae have been imaged to identify the soft tissue structures involved in the hearing process. The present experimental approach was essential for visualizing the inner ear structures involved in the hearing process in an intact cochlea.     

Differential phase contrast in scanning optical microscopy

High-quality high-resolution transmission and reflection images produced using a scanning optical microscope and the split-detector technique are presented. These images exhibit differential phase contrast, the method avoiding some drawbacks of the usual Nomarski DIC arrangement. Imaging is treated theoretically and compared with the Nomarski method.     

Confocal differential interference contrast (DIC) microscopy: including a theoretical analysis of conventional and confocal DIC imaging

A technique for obtaining differential interference contrast (DIC) imaging using a confocal microscope system is examined and its features compared to those of existing confocal differential phase contrast (DPC) techniques as well as to conventional Nomarski DIC. A theoretical treatment of DIC imaging is presented, which takes into account the vignetting effect caused by the finite size of the lens pupils. This facilitates the making of quantitative measurements in DIC and allows the user to identify and select the most appropriate system parameters, such as the bias retardation and lateral shear of the Wollaston prism.     

利用Monte Carlo模拟技术研究OCT图像对比度

建立了光在生物组织中传播的模型,利用MonteCarlo模拟技术快速计算和可移植性的优点,研究了OCT图像对比度与显微物镜数值孔径、焦距深度、时间门参数的关系,并给出它们的关系曲线.通过在所建立的模型基础上加入透镜透过率函数和光学传递函数,弥补了以往程序只能模拟光在生物组织中传播行为的缺点.该模型不仅可以分析生物组织图像与生物光学特性的关系,而且还可以指导OCT结构的完善和创新.模拟结果表明:在构建OCT时,参考臂与样品臂的这两个显微物镜的数值孔径越大,生物组织的采样深度越浅,处理信号的时间门宽度越小(但时间门宽度不能小于激光脉冲时间),混浊生物组织图像对比度越好.     

荧光成像系统对比度分析与成像仿真

目前荧光成像技术在生物医学领域得到越来越广泛的应用。为了缩短产品设计和开发周期,且能更直观地反映系统的成像效果,根据各模块光谱特性曲线,提出了荧光成像链路模型。利用该模型,对荧光成像系统的对比度进行了分析,验证了滤光片光密度(OD)值的合理范围是在5~7之间。最后以荧光显微镜为例,对系统成像过程进行了仿真。结果表明,各模块不匹配也会对系统对比度产生影响,仿真图像能够直观反映出系统的匹配程度和成像效果,并与实际系统测试结果相吻合,证明了该链路模型仿真的可行性和有效性。     

Contrast in confocal scanning microscopy with a finite detector

The optical properties of a general scanning microscope are determined within the framework of Fourier imaging theory. For a simple model optical system, with Gaussian lens and detector apertures, the contrast transfer function can be expressed in terms of elementary functions. The theory predicts that spatial resolution and depth discrimination vary continuously with detector aperture and that defocus phase contrast is present in transmission images obtained with a symmetric objective, collector lens confocal microscope.     

Fibre-optic nonlinear optical microscopy and endoscopy

Nonlinear optical microscopy has been an indispensable laboratory tool of high‐resolution imaging in thick tissue and live animals. Rapid developments of fibre‐optic components in terms of growing functionality and decreasing size provide enormous opportunities for innovations in nonlinear optical microscopy. Fibre‐based nonlinear optical endoscopy is the sole instrumentation to permit the cellular imaging within hollow tissue tracts or solid organs that are inaccessible to a conventional optical microscope. This article reviews the current development of fibre‐optic nonlinear optical microscopy and endoscopy, which includes crucial technologies for miniaturized nonlinear optical microscopy and their embodiments of endoscopic systems. A particular attention is given to several classes of photonic crystal fibres that have been applied to nonlinear optical microscopy due to their unique properties for ultrashort pulse delivery and signal collection. Furthermore, fibre‐optic nonlinear optical imaging systems can be classified into portable microscopes suitable for imaging behaving animals, rigid endoscopes that allow for deep tissue imaging with minimally invasive manners, and flexible endoscopes enabling imaging of internal organs. Fibre‐optic nonlinear optical endoscopy is coming of age and a paradigm shift leading to optical microscope tools for early cancer detection and minimally invasive surgery.     

Immersion oil for high‐resolution live‐cell imaging at 37°C: optical and physical characteristics

The use of normal immersion oil, developed for 23°C, at 37°C greatly compromises both axial resolution and signal intensity. We developed and characterized an immersion oil for optimal performance in live‐cell imaging at 37°C. We quantify the improvements in resolution and intensity obtained when using the new oil instead of its standard 23°C counterparts.     

Possibility of charge contrast imaging of polymeric materials

Preliminary results illustrate the possibility of charge contrast imaging (CCI) of polymeric materials. Possible CCI images of low-density polyethylene and polyvinyl chloride reveal details that may aid in the characterization of the microstructure of polymeric materials. These pictures were obtained with a Hitachi S-3000N variable pressure scanning electron microscope with the environmental secondary electron detector (ESED).     

Collection deficiencies of scanning electron microscopy signal contrasts measured and corrected by differential hysteresis image processing

Limitations of scanning electron microscopy (SEM) image resolution and quality were measured in digital image data and their effect on image contrasts was analyzed and corrected by differential hysteresis (DH) processing. DH processing is a mathematical procedure that utilizes hysteresis properties of intensity variations in the image for a segmentation of differential contrast patterns. These patterns display contrast properties of the data as coherent full-frame images. The contrast segmentation is revertible so that the original image can be restored from the sum of the sequentially extracted DH contrast patterns. DH imaging enhances weak contrast components so that they are more easily recognizable and displays SEM image data free of signal collection efficiency contrasts. Example image data include environmental SEM (ESEM) and SEM images of low and mediumhigh magnifications where collection deficiencies included charging of the specimen surface, obstructions from specimen topography, and uneven signal collection properties of the detector. ESEM low-vacuum image data, which appear to be of high quality, contained local areas of reduced contrasts due to residual surface charging. In such areas, signal contrasts were reduced up to 80%, which suppressed most of the weak short-range contrasts. In low-magnification SEM images, up to 93% of the local high precision contrast was lost from the various adverse effects which diminished the pixel-related contrast resolution of the microscope and resulted in images with low detail. Also, at medium magnification, surface charging effects dramatically reduced the image quality because contrasts resulting from local electron beam/specimen interactions were reduced by as much as 71%. DH imaging restored the local contrast losses by elimination of the collected distorted fraction of signal contrasts and reconstitution of the collected maintained fraction. Restored DH images are of superior quality and enhance the imaging capability of the conventional SEM. DH contrast segmentation provides an improved basis for the measurement of various signal contrast components and detector performances. The DH analysis will ultimately facilitate a precise deduction of specimen properties from extracted contrast patterns.     

TEM in materials science—past,present and future

The progress in transmission electron microscopy as applied in materials science is reviewed briefly, from the era of replica techniques in the 1940s and 1950s, through the development of the diffraction contrast technique in the late 1950s and 1960s, to the present day when instrumental resolution is sufficient to obtain structure images of a wide variety of crystalline solids. One of the most important advances has been the development of combined imaging and microanalytical techniques in a single instrument. The versatility of the TEM technique with atomic resolution and microanalytical facilities, the variety of signals and contrast effects available, and its universal application, establishes it as an essential tool in materials science for the foreseeable future.     

Axial Phase-Darkfield-Contrast (APDC), a new technique for variable optical contrasting in light microscopy

Axial phase-darkfield-contrast (APDC) has been developed as an illumination technique in light microscopy which promises significant improvements and a higher variability in imaging of several transparent 'problem specimens'. With this method, a phase contrast image is optically superimposed on an axial darkfield image so that a partial image based on the principal zeroth order maximum (phase contrast) interferes with an image, which is based on the secondary maxima (axial darkfield). The background brightness and character of the resulting image can be continuously modulated from a phase contrast-dominated to a darkfield-dominated character. In order to achieve this illumination mode, normal objectives for phase contrast have to be fitted with an additional central light stopper needed for axial (central) darkfield illumination. In corresponding condenser light masks, a small perforation has to be added in the centre of the phase contrast providing light annulus. These light modulating elements are properly aligned when the central perforation is congruent with the objective's light stop and the light annulus is conjugate with the phase ring. The breadth of the condenser light annulus and thus the intensity of the phase contrast partial image can be regulated with the aperture diaphragm. Additional contrast effects can be achieved when both illuminating light components are filtered at different colours. In this technique, the axial resolution (depth of field) is significantly enhanced and the specimen's three-dimensional appearance is accentuated with improved clarity as well as fine details at the given resolution limit. Typical artefacts associated with phase contrast and darkfield illumination are reduced in our methods.     

Real‐time three‐dimensional imaging of cell division by differential interference contrast microscopy

Differential interference contrast (DIC) microscopy can provide information about subcellular components and organelles inside living cells. Applicability to date, however, has been limited to 2D imaging. Unfortunately, understanding of cellular dynamics is difficult to extract from these single optical sections. We demonstrate here that 3D differential interference contrast microscopy has sub‐diffraction limit resolution both laterally and vertically, and can be used for following Madin Darby canine kidney cell division process in real time. This is made possible by optimization of the microscope optics and by incorporating computer‐controlled vertical scanning of the microscope stage.