Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation

The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.     

数字化图像技术测量凝胶电泳蛋白质含量的三种方法比较

本文基于光密度测量原理,在考虑了凝胶电泳图像数字化过程中照射光源强度、凝胶背景、摄像机等参量对蛋白质含量计算结果的影响后,提出了凝胶电泳蛋白定量测量的改进公式和实验方法,并与目前使用较多的灰度积分法、灰度积分拟合法进行了实验比较。结果显示,改进公式所得结果更接近真实值、标准差更小、且不受凝胶背景变化的影响。     

Contributions to the contrast in experimental high-angle annular dark-field images

This paper reports on a study of the contributions to the image contrast of high-angle annular dark field (HAADF) images acquired in scanning transmission electron microscopy. Experimental HAADF images were obtained from a model system consisting of an epitaxial perovskite PbTiO3 film grown on a SrTiO3 single crystal. This sample allowed for the study of the intensities of a wide range of atomic numbers. The main objective of the paper was to quantify the influence of TEM foil thickness on the image contrast, but the effects of the annular detector inner angle and the probe forming lens focus were also studied. Sample thicknesses ranging from approximately 10 nm to more than 400 nm were investigated. The image contrast was relatively insensitive to changes in inner angle. The main impact of sample thickness was a rapid increase in a background intensity that contributed equally to the intensities of the atomic columns and the channels between them. The background intensity and its increase with thickness reflected the average atomic number of the crystal. Subtraction of the background intensity allowed for a quantitative interpretation of image contrast in terms of atomic numbers and comparison with multislice image simulations. The consequences for the analysis of interfaces in terms of atom column occupancies are discussed.     

Essential experimental parameters for quantitative structure analysis using spherical aberration-corrected HAADF-STEM

The accuracy of quantitative analysis for Z-contrast images with a spherical aberration (C_s) corrected high-angle annular dark-field (HAADF) scanning transmission electron microscope (STEM) using SrTiO₃(0 0 1) was systematically investigated. Atomic column and background intensities were measured accurately from the experimental HAADF-STEM images obtained under exact experimental condition. We examined atomic intensity ratio dependence on experimental conditions such as defocus, convergent semi-angles, specimen thicknesses and digitalized STEM image acquisition system: brightness and contrast. In order to carry out quantitative analysis of C_s-corrected HAADF-STEM, it is essential to determine defocus, to measure specimen thickness and to fix setting of brightness, contrast and probe current. To confirm the validity and accuracy of the experimental results, we compared experimental and HAADF-STEM calculations based on the Bloch wave method.     

Computation of contrasts in atomic resolution electron spectroscopic images of planar defects in crystalline specimens

The image obtained in a conventional transmission electron microscope contains contributions from elastically and from inelastically scattered electrons. The electron spectroscopic imaging mode of an energy-filtering transmission electron microscope allows us to separate these two different contributions by inserting an energy-selecting slit in the energy-dispersive plane of an imaging energy filter. Selecting a specific energy loss corresponding to the ionization of the inner shell of a particular element one can obtain information on the distribution of the element within the specimen. The contrast is then caused by inelastically scattered electrons. For crystalline specimens, however, the contrast will be influenced additionally by the elastic contrast. This elastic contrast arises from electron diffraction and increases with increasing crystal thickness. Therefore the intensity distribution in the image cannot directly be interpreted as an elemental map. For a reliable interpretation of contrast formation in elemental maps it is therefore necessary to compute theoretical energy-loss images for various crystal thicknesses and compare these images with the experimental images. As an example we discuss the influence of electron diffraction effects on energy-loss images of two crystals with planar defects. Linescans are computed for various thicknesses of these crystals. Our calculations are performed using first-order perturbation theory to describe the transitions between the Bloch-wave states of the incident electron. The computed linescans for various crystal thicknesses show clearly that the influence of the elastic contrast on an image increases when we investigate thicker specimens. Furthermore, the comparison between elastic and energy-loss images demonstrates the partial preservation of the elastic contrast as a function of thickness. We find that for specimens thicker than about one third of the extinction length (here approximately 80-100 A) it is impossible to interpret an energy-loss image directly as elemental map.     

Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated

Video-enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video-enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video-enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced differential interference contrast. By carrying out background subtraction with 12-bit images, the number of grey levels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion-enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.     

基于元胞自动机的动态背景运动目标检测

针对传统运动目标检测算法在动态背景条件下难以准确检测出运动目标的问题,提出了一种基于元胞自动机的动态背景运动目标检测算法。首先,根据SLIC算法分割视频图像,并应用多模态混合动态纹理模型对视频图像进行背景建模。然后,融合空时显著性检测与基于元胞自动机的自动更新机制得到优化的显著性图。最后,通过对优化后的显著性图做适当的阈值分割处理得到视频图像中的运动目标。实验仿真结果表明,在动态背景条件下该算法可以有效的抑制视频图像中非运动目标的显著性物体对检测结果带来的影响,检测运动目标的精度较高,并且具有一定的鲁棒性。     

Multichannel high-performance optoelectronic control of the surface of fuel pellets

Optoelectronic methods for controlling the appearance of nuclear reactor fuel pellets are considered. In the proposed methods, reflected images of pellet surfaces are taken by digital cameras, which provides high contrast of defective areas against the frame background. Image processing algorithms for identification of defective products are given. An experimental model of a system with a capacity of up to 10 pellets per second was designed. A database containing surface images of simulators and real pellets was designed to train the system. Results of real time processing of the obtained images show that the probability of detection of defective pellets is not less than 95%.     

Scanning probe microscopies applied to the study of the domain wall in a ferroelectric crystal

Scanning near‐field optical microscopy is capable of measuring the topography and optical signals at the same time. This fact makes this technique a valuable tool in the study of materials at nanometric scale and, in particular, of ferroelectric materials, as it permits the study of their domains structure without the need of chemical etching and, therefore, not damaging the surface (as will be demonstrated later). We have measured the scanning near‐field optical microscopy transmission, as well as the topography, of an RbTiOPO₄ single crystalline slab, which exhibits two different of macroscopic ferroelectric domains. A chemical selective etching has been performed to distinguish between them, obtaining areas with a noticeable roughness (C⁻ domain) in comparison with the original flat aspect of the other ones (C⁺ domain). The effects of the selective chemical etching have been quantified in topographic images obtained by means of our fibre tip probe, and have been compared to topographic images obtained by Atomic Force Microscopy, with a better resolution. The near‐field optical transmission images recorded have been obtained under different excitation wavelengths. These images are modulated by the light scattering due to the grains at the rough surface, which depends on the excitation wavelength used. In addition, they show a significant optical contrast due to the variations of the dielectric constant on the proximity of the ferroelectric domain wall.     

Quantification of high-resolution electron microscope images of amorphous carbon

Quantitative comparisons of experimentally obtained and simulated high-resolution electron microscope images have shown that the contrast in experimental images is usually much less than is predicted by simulations. The aim here is to investigate this loss of contrast as a function of image spatial frequency using high-resolution images of amorphous carbon. It seems that experimental images of amorphous carbon have an unexpectedly high contrast for the low spatial frequencies but that the loss of contrast is constant for frequencies above 0.5 nm(-1).     

Reflection electron microscopy methods for the study of surface structure

The techniques of reflection electron microscopy (REM) using TEM instruments and scanning reflection electron microscopy (SREM) using STEM instruments have been explored as means for the observation of surface structure with high spatial resolution, better than 1 nm in each case. Under the ordinary environment of a commercial TEM instrument, we have studied the contrast in REM images of atomic steps and made comparison with the calculated results from the multi-slice dynamical diffraction theory. Comparison has also been made between the REM images of defects and the calculated images based on the column approximation. The influence of surface resonances on the contrast has been investigated. By SREM performed in a modified HB5 STEM with attached high vacuum preparation chamber, we have observed the formation of periodically distributed Pd particles on the surface of cleaved MgO.     

基于超声造影剂谐波图像的流场测量技术

本文研究了基于超声造影剂的血流运动场估计与显示技术,通过在血液中注入超声造影剂作为示踪粒子,对造影谐波图像进行时域相关处理,可得到成像部位的二维流场分布图。相对于常规Ｄｏｐｐｌｅｒ方法中用Ｄｏｐｐｌｅｒ回波信号的频偏计算流速值,该技术可直接从超声图像提取与夹角无关的流速矢量信息。本文通过流动模型验证该方法,浸入超声水槽中的乳胶管中流动着血液替代品,沿水流方向进行超声成像,对实验所得的造影后的Ｂ超图像以及谐波图像,用一种节省计算量的多尺度相关算法进行处理,并相互比较。结果显示,谐波图像相对Ｂ超基波图像具有更高的信杂比,从根本上解决了基波图像低信噪比对时域相关测量精度的限制,可以得到与夹角无关的二维血流场分布图,该方法是医学和有关工业领域中超声流场测量的一种有效的方法。     

Automatic setae segmentation from Chaetoceros microscopic images

A novel image processing model Grayscale Surface Direction Angle Model (GSDAM) is presented and the algorithm based on GSDAM is developed to segment setae from Chaetoceros microscopic images. The proposed model combines the setae characteristics of the microscopic images with the spatial analysis of image grayscale surface to detect and segment the direction thin and long setae from the low contrast background as well as noise which may make the commonly used segmentation methods invalid. The experimental results show that our algorithm based on GSDAM outperforms the boundary‐based and region‐based segmentation methods Canny edge detector, iterative threshold selection, Otsu's thresholding, minimum error thresholding, K‐means clustering, and marker‐controlled watershed on the setae segmentation more accurately and completely. Microsc. Res. Tech. 77:684–690, 2014. © 2014 Wiley Periodicals, Inc.     

Elemental mapping at the atomic scale using low accelerating voltages

Atomic resolved elemental mapping is demonstrated at 80 keV with an aberration-corrected scanning transmission electron microscope on specimens of SrTiO₃ and BaTiO₃/SrTiO₃. The maps were acquired with acquisition times as short as 30 ms per pixel (limited by the spectrometer speed), and show very high signal-to-noise ratio and very good detection limits. The features in the elemental maps are interpreted with the help of elastic–inelastic multislice calculations, which show good agreement with experimental images. The elemental maps of Ti, Sr and Ba and their contrast at the interface between BaTiO₃ and SrTiO₃ are discussed, following a comparison with calculations, assuming an atomically sharp interface. The features in the energy-filtered maps and the background intensities, and the influence of the energy position of the integration windows are discussed in terms of the origins of the signals and the features with respect to the details shown in the high-angle annular dark-field images. The benefits of elemental mapping at 80 keV as compared to 200 keV are also discussed in terms of electron beam damage. Finally, applications of elemental mapping to the detection of La atoms in solid solution in Ba_3.25La_0.75Ti₃O₁₂ films are also shown.     

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.     

Algorithms for Bayesian background-subtracted Fourier darkfield imaging.

Formal consideration of prior information on the Fourier amplitude of background contrast in an image, using the same Bayesian principles of statistical inference which underlie thermodynamics, allows one to subtract background without favoring only selected parts of frequency space. Without the bias in frequency space which causes periodicity bleeding and mars literal interpretation of Fourier-filtered images, the shape transform of aperiodic objects can be left intact. Algorithms for Bayesian background subtraction from one- and two-dimensional images are presented which further consider, in ad hoc fashion, one's uncertainty about background amplitude. The results help explain the reported success of Fourier truncation, and indicate that Bayesian background-subtracted images can minimize root-mean-square image error, as well as periodicity bleeding, in comparison to Fourier-filtered and Fourier-truncated alternatives.     

利用光谱滤波技术提高电视跟踪系统作用距离的研究

详细分析了目标、天空背景的光谱辐射特性、大气条件以及电视跟踪系统各环节对探测靶面上目标与背景对比度的影响,确定了利用光谱滤波提高电视跟踪系统作用距离的理论基础。实测了作为跟踪目标的3号曳光弹的光谱辐射特性;根据与电视跟踪系统工作环境相近的大气模式,从理论上计算、分析了天空背景的光谱辐射特性。通过计算机模拟与实测各种滤光片对天空背景辐射的抑制效果及对模拟目标信号衰减程度相结合的办法,从理论及实验上研究了光谱滤波提高电视跟踪系统作用距离的规律。并且从光谱滤波角度提出了对系统所采用的探测器及合作目标信号的某些特性的改进意见。对九种不同背景条件下光谱滤波效果的计算机模拟分析表明:光谱滤波能稳定有效地提高探测器靶面上的目标与背景的对比度。对3号曳光弹,在海平面45km,水平路径上.在0.810μm,处的窄带光谱滤波能使探测器靶面上的对比度提高4～7倍,作用距离可由40km提高到(50～53)km。实验表明:光谱滤波后,本征对比度可提高1.25～3.59倍。     

Electron phase microscopy at 1 MeV with graphite crystal supports

Thin phase objects can be seen with good contrast with 1 MeV electrons if they are supported on a graphite crystal approximately half an extinction distance thick and viewed near 101 or 112 bend contours. The shape of unstained tobacco mosaic virus is easily seen with crystal phase contrast although it is almost invisible in a normal bright field micrograph taken with 1 MeV electrons. Phase contrast images of both TMV and 109 nm diameter latex spheres are observed to be bright against a grey background on one side of a 101 or 112 bend contour and dark against a grey background on the other side of the bend contour. For strong contrast the crystal diffraction vector g must have a component parallel to the beam. Contours such as 100 and 110 do not give strong phase contrast. The phase contrast images for biological material gradually increase in contrast as the specimens are irradiated by the electron beam. An electron exposure of about 10⁴ electrons nm^?2 is required to obtain maximum contrast.     

焊接缺陷的X射线自动检测图像处理

X射线无损检测对于焊接结构件的质量保证具有重要意义。使用Image J图像处理软件和Java语言开发插件,对焊接点中气孔分布以及面积等缺陷参数进行自动检测并对获得的图像进行分析处理。提出利用区域标记法分割出有效分析区域,先去除无效区域后再对感兴趣区域进行气孔提取,相较于传统的全局图像差分法,可以减少背景对缺陷检测的影响,降低误判率。设计出的检测方法能够对特定图像进行批量分析,可以减少检测的时间。     

Nonlinear imaging using annular dark field TEM

Annular dark field TEM images exhibit a dominant mass-thickness contrast that can be quantified to extract single atom scattering cross sections. On top of this incoherent background, additional lattice fringes appear with a nonlinear information limit of 1.2A at 150 kV. The formation of these fringes is described by coherent nonlinear imaging theory and good agreement is found between experimental and simulated images. Calculations furthermore predict that the use of aberration corrected microscopes will improve the image quality dramatically.