Quantitative phase-amplitude microscopy II: differential interference contrast imaging for biological TEM

Although phase contrast microscopy is widespread in optical microscopy, it has not been as widely adopted in transmission electron microscopy (TEM), which has therefore to a large extent relied on staining techniques to yield sufficient contrast. Those methods of phase contrast that are used in biological electron microscopy have been limited by factors such as the need for small phase shifts in very thin samples, the requirement for difficult experimental conditions, or the use of complex data analysis methods. We here demonstrate a simple method for quantitative TEM phase microscopy that is suitable for large phase shifts and requires only two images. We present a TEM phase image of unstained Radula sp. (liverwort spore). We show how the image may be transformed into the differential interference contrast image format familiar from optical microscopy. The phase images contain features not visible with the other imaging modalities. The resulting technique should permit phase contrast TEM to be performed almost as readily as phase contrast optical microscopy.     

Quantitative phase-amplitude microscopy. III. The effects of noise

We explore the effect of noise on images obtained using quantitative phase‐amplitude microscopy – a new microscopy technique based on the determination of phase from the intensity evolution of propagating radiation. We compare the predictions with experimental results and also propose an approach that allows good‐quality quantitative phase retrieval to be obtained even for very noisy data.     

Quantitative X-ray projection microscopy: phase-contrast and multi-spectral imaging

We outline a new approach to X‐ray projection microscopy in a scanning electron microscope (SEM), which exploits phase contrast to boost the quality and information content of images. These developments have been made possible by the combination of a high‐brightness field‐emission gun (FEG)‐based SEM, direct detection CCD technology and new phase retrieval algorithms. Using this approach we have been able to obtain spatial resolution of < 0.2 µm and have demonstrated novel features such as: (i) phase‐contrast enhanced visibility of high spatial frequency image features (e.g. edges and boundaries) over a wide energy range; (ii) energy‐resolved imaging to simultaneously produce multiple quasi‐monochromatic images using broad‐band polychromatic illumination; (iii) easy implementation of microtomography; (iv) rapid and robust phase/amplitude‐retrieval algorithms to enable new real‐time and quantitative modes of microscopic imaging. These algorithms can also be applied successfully to recover object–plane information from intermediate‐field images, unlocking the potentially greater contrast and resolution of the intermediate‐field regime. Widespread applications are envisaged for fields such as materials science, biological and biomedical research and microelectronics device inspection. Some illustrative examples are presented. The quantitative methods described here are also very relevant to projection microscopy using other sources of radiation, such as visible light and electrons.     

A simple method allowing DIC imaging in conjunction with confocal microscopy

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique ( Barone-Nugent et al., 2002 ). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.     

Quantitative low temperature optical microscopy of biological systems

A comprehensive review is presented of low temperature optical microscopy techniques as applied to the study of freezing processes in biological systems. Emphasis is placed on analysis of physical and physiological parameters which were measured and/or controlled and the procedures for effecting such operations. Quantitative analysis of photomicrographs by digital computer processing is also discussed.     

偏振相移技术在DIC显微术中的应用研究

微分干涉（DIC）显微技术使用诺曼斯基棱镜完成光束的分割、合成、最后发生干涉,将样品上各个部分折射率、厚度的变化率或表面起伏的不同转化为像面上光强的差别,一般情况下,无法进行定量测量。文章将激光偏振相移技术应用于DIC显微术中,建立并推导出光学数学模型。该模型利用无限远光学系统优化系统光路,使系统减小了因为使用激光光源、     

Multimodal microscopy by digital image processing

A matching algorithm is proposed for aligning microscope images obtained using different modalities, making use of cross-correlations of outputs from Prewitt's edge filter. Brightfield, phase contrast and differential interference contrast microscope images of algal and bacterial cells from an experimental, high-rate algal pond are used for illustration. The information content of multimodal images is explored using principal components analysis and colour displays, and an image which represents optical thickness is constructed digitally.     

微分相衬干涉显微镜定量测量表面形貌

改进了用于定量测量样品表面形貌的微分相衬干涉显微镜系统,对系统中由Nomarski棱镜引起的相位差β的消零工作提出了一种新的方法,实验证明是有效可靠的,并且对样品表面三维形貌重构、截面轮廓比对及系统的测量精度进行了实验研究.     

机器视觉精密测量中的显微光学聚焦

考虑显微光学涉及的聚焦精度对机器视觉精密测量效果的影响,开展了显微视觉环境下对图像聚焦技术综合定量评价的研究。建立了偏移率等系列性能指标,对13组清晰度函数在显微视觉条件下的无偏性、单峰性、分辨力等进行了综合评价,优选出方差函数和Brenner函数分别用于粗聚焦和精聚焦阶段的清晰度计算。建立了分步爬山搜索法,实现了显微自动聚焦。与传统爬山法相比,提出的方法聚焦时间显著缩短,重复精度提高约24%。将建立的自动聚焦与图像测量方法应用于某电液伺服阀衔铁气隙测量中,得到的测量均值与工具显微镜结果相近,而测量标准差可达1.9μm,测量效率也显著提高。最后对伺服阀加电条件下的气隙动力学特性进行了测试,获得了驱动电流-衔铁气隙之间的关系,为在线装配/装调提供了重要依据。     

Quantitative analysis of backscattered-electron contrast in scanning electron microscopy

Backscattered-electron scanning electron microscopy (BSE-SEM) imaging is a valuable technique for materials characterisation because it provides information about the homogeneity of the material in the analysed specimen and is therefore an important technique in modern electron microscopy. However, the information contained in BSE-SEM images is up to now rarely quantitatively evaluated. The main challenge of quantitative BSE-SEM imaging is to relate the measured BSE intensity to the backscattering coefficient η and the (average) atomic number Z to derive chemical information from the BSE-SEM image. We propose a quantitative BSE-SEM method, which is based on the comparison of Monte–Carlo (MC) simulated and measured BSE intensities acquired from wedge-shaped electron-transparent specimens with known thickness profile. The new method also includes measures to improve and validate the agreement of the MC simulations with experimental data. Two different challenging samples (ZnS/Zn(O_xS_1–x)/ZnO/Si-multilayer and PTB7/PC₇₁BM-multilayer systems) are quantitatively analysed, which demonstrates the validity of the proposed method and emphasises the importance of realistic MC simulations for quantitative BSE-SEM analysis. Moreover, MC simulations can be used to optimise the imaging parameters (electron energy, detection-angle range) in advance to avoid tedious experimental trial and error optimisation. Under optimised imaging conditions pre-determined by MC simulations, the BSE-SEM technique is capable of distinguishing materials with small composition differences.     

基于微分干涉相衬的相位分析法研究

通过对微分干涉相衬显微定量测量方法进行研究,提出了一种更有效的相位分析法。即在不对双光束干涉光路进行改造或处理的前提下,通过对光学成像进行处理而得到理想的结果。即把图像中的光强信号转变成相位信号,并通过维纳滤波对噪声进行了消除,最后获得表面微观形貌定量参数。     

Precision of light intensity measurement in biological optical microscopy

Standardization and calibration of optical microscopy systems have become an important issue owing to the increasing role of biological imaging in high‐content screening technology. The proper interpretation of data from high‐content screening imaging experiments requires detailed information about the capabilities of the systems, including their available dynamic range, sensitivity and noise. Currently available techniques for calibration and standardization of digital microscopes commonly used in cell biology laboratories provide an estimation of stability and measurement precision (noise) of an imaging system at a single level of signal intensity. In addition, only the total noise level, not its characteristics (spectrum), is measured. We propose a novel technique for estimation of temporal variability of signal and noise in microscopic imaging. The method requires registration of a time series of images of any stationary biological specimen. The subsequent analysis involves a multi‐step process, which separates monotonic, periodic and random components of every pixel intensity change in time. The technique allows simultaneous determination of dark, photonic and multiplicative components of noise present in biological measurements. Consequently, a respective confidence interval (noise level) is obtained for each level of signal. The technique is validated using test sets of biological images with known signal and noise characteristics. The method is also applied to assess uncertainty of measurement obtained with two CCD cameras in a wide‐field microscope.     

Quantitative theory of ideal phase-contrast microscopy,taking object width into account

In ‘ideal’ phase-contrast microscopy all the direct light and none of the diffracted light is influenced by the phase plate in the back focal plane of the objective. Contrary to almost all previous work, it appears that the intensity of an ideal phase-contrast image is affected not only by the transmittance and retardation of the object and of the phase plate, but also by the width of the specimen (or total width of multiple specimens) relative to the microscopic field. Equations and computer code are presented with which the intensity of such images can be calculated. Previously published equations are special cases, and implicitly or explictly assume either that the object is of negligible width, or occupies precisely half the microscopic field. The absolute brightness of an image in ideal central dark-field microscopy is a function of the object retardation, but the intensity of the image relative to the background is a function only of the width of the object(s) relative to the field. The equations give results for ideal phase-contrast microscopy identical with those of a computer program simulating microscopic imaging. The program can in addition take into account non-ideal factors including a finite width of phase plate, finite objective aperture, deviations from best focus, glare, primary spherical aberration and obliquity of the coherent illumination.     

Fluorescence scanning near-field optical microscopy of polyfluorene composites

Fluorescence scanning near-field optical microscopy (SNOM) is used to investigate binary polyfluorene-based composites of varying composition. The samples investigated contain blends of the polymer poly(9,9'-dioctylfluorene-cobenzothiadiazole), F8BT, with similar polyfluorenes of wider band gap. Images acquired from a film containing 50% by weight F8BT exhibit a high degree of correlation between the topography and fluorescence, with an F8BT-rich phase which protrudes from the surface of the film forming isolated regions with sizes from hundreds of nanometres to several micrometres. A film containing 10% by weight F8BT also has micrometre-size F8BT-rich regions, but also present are small and locally varying proportions of F8BT in the other polyfluorene component phase, indicating a hierarchy of phases within this sample. The fluorescence and topographic images of a third sample studied, containing 90% by weight F8BT, display no correlation, demonstrating that it is not always appropriate to use topographic information to determine the phase structure within polymer blends. The fluorescence SNOM images acquired from these samples are able to assist our understanding of the photovoltaic efficiency of devices fabricated from these films, which are governed by the extent of the interfacial area between these two constituent polymers.     

Quantitative high resolution transmission electron microscopy of nanostructured semiconductors

Peak‐finding procedures and the geometric phase method of quantitative high resolution electron microscopy (qHRTEM) were applied to determine the local strain and the chemical composition of nanostructured semiconductor materials. The growth of the structures investigated was induced by minimization of strain energy. The analysis of strain distribution is necessary for the understanding of the self‐organized formation of nanostructures. The possibilities and limitations of the methods are discussed in detail by analysing HRTEM images of (Si,Ge) islands and of a double layer of stacked quantum dots of (In,Ga)As and Ga(Sb,As).     

Comparison of holotomographic microscopy and coherence-controlled holographic microscopy

Quantitative phase imaging (QPI) is a powerful tool for label-free visualisation of living cells. Here, we compare two QPI microscopes – the Telight Q-Phase microscope and the Nanolive 3D Cell Explorer-fluo microscope. Both systems provide unbiased information about cell morphology, such as individual cell dry mass, perimeter and area. The Q-Phase microscope uses artefact-free, coherence-controlled holographic imaging technology to visualise cells in real time with minimal phototoxicity. The 3D Cell Explorer-fluo employs laser-based holotomography to reconstruct 3D images of living cells, visualising their internal structures and dynamics. Here, we analysed the strengths and limitations of both microscopes when examining two morphologically distinct cell lines – the cuboidal epithelial MDCK cells which form multicellular clusters and solitary growing Rat2 fibroblasts. We focus mainly on the ability of the devices to generate images suitable for single-cell segmentation by the built-in software, and we discuss the segmentation results and quantitative data generated from the segmented images. We show that both microscopes offer slightly different advantages, and the choice between them depends on the specific requirements and goals of the user.     

传统光学显微镜与近场光学显微镜

近场光学显微镜是对于常规光学显微镜的革命。它不用光学透镜成像 ,而用探针的针尖在样品表面上方扫描获得样品表面的信息。分析了传统光学显微镜与近场光学显微镜成像原理的物理本质和两种显微镜系统结构的异同点。介绍了光纤探针的制作方法。重点讨论了近场探测原理、光学隧道效应及非辐射场的性质     

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.     

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.     

Evanescent-wave microscopy: A simple optical configuration

Under suitable conditions, the region of the aqueous phase immediately adjacent to a glass-water interface can be selectively illuminated using the evanescent wave created when total internal reflection occurs at the interface. Objects in the aqueous phase away from the glass become effectively invisible, since the intensity of the evanescent wave decays exponentially with distance from the interface. Previous methods of generating evanescent waves for light microscopy have employed accessory light sources and optical components that directed the illumination on the specimen from one side. The asymmetric illumination creates a surprising orientation dependence of visibility for some specimens. Objects such as microtubules are totally invisible unless they are orientated nearly perpendicular to the direction of illumination. An explanation of this phenomenon is provided in terms of the geometry of diffraction of light by long thin objects. A simple method of achieving evanescent-wave illumination is described and shown to be useful in practice for biological specimens. In contrast to previously described methods, the present arrangement has the advantage of producing circularly symmetric illumination, and of utilizing only standard optical microscope components. The system has been used for imaging specimens both by light scattering and by fluorescence. It has proved useful for following the fragmentation of flagella into isolated microtubules, observing microtubules gliding over dynein adsorbed to a surface, and also for determining the arrangement of vinculin and alpha-actinin in cell-substratum attachment sites and termini of growing myofibrils in cardiac cells.