Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification

Light microscopic autoradiographs of H-thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase-contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.     

Application of ion etching to immunoscanning electron microscopy

A method for achieving both the light and electron microscopic observations of the same immunolabeled semithin section is described. Mild ion etching (IE) was performed on the semithin LR white resin sections of rat pancreas to evaluate conditions for scanning electron microscopic secondary electron image observations. Before immunocytochemical staining, very mild, rapid etching was conducted as follows: ionization voltage 300 V, operating vacuum 35 Pa, and etching time 1 min, employing an ion coater above sections on glass slides. The sections were immunohistochemically stained with anti-insulin and immunogold in association with silver enhancement techniques for light microscopic observation, in which B cells in pancreatic islets were positively stained brown. Subsequently, essential mild IE was performed over the stained section as follows: 350 V, 38 Pa, 29 min. The samples were coated with platinum for scanning electron microscopic secondary electron images, in which the cores of secretory granules of the B cells were positively labeled with gold-silver particles. The present method is suitable for detection of substances involving immunogold labeling. It enables us to obtain high-resolution images at low magnification that can be correlated with light microscopic observations. Middle to high magnifications are applicable for detailed observations with secondary electron imaging scanning electron microscopy.     

铁谱磨粒图像多焦点融合算法研究

铁谱分析技术是一种常用的磨损监测技术。受限于高倍物镜下的景深限制,一张铁谱大磨粒图像往往只有局部聚焦清晰的特征。为了能够解决在高倍物镜下铁谱大磨粒图像的自动化清晰采集以及高质量图像融合问题,设计并构建一套自动化扫描显微系统,该系统可进行多焦点铁谱图像的自动扫描采集;同时,提出一种基于相位一致性的铁谱磨粒图像多焦点融合算法,对自动扫描的多焦点图像进行融合,得到清晰的磨粒图像。实验结果表明,设计的自动化扫描显微系统能快速完成多焦点铁谱图像的自动化采集流程,提出的图像融合算法相较于传统的小波图像融合算法具有更高的图像评价质量,并能获得更加清晰的图像边缘信息。     

Restoration of distorted colour microscopic images from transverse chromatic aberration of imperfect lenses

An algorithm is presented for restoration of colour microscopic images with distortions from imperfect microscope lenses having transverse chromatic aberrations, resulting in a magnification that slightly varies with wavelengths or colours. The differential of each colour component image is computed as the difference between the component image and its slightly magnified version. The absolute values in the differential component images are generally higher at the edges where greater discontinuities occur. The two cross-correlation functions of the absolute differentials between red and green colours and between red and blue colours are then computed. The maximum in the two cross-correlation functions were sought, respectively, and the cross-correlation delays were then calculated. The two cross-correlation delays were used to determine dispersions and to realign the three colour components. Results of real microscopic images are provided. The restored image and the original are compared both visually and quantitatively in terms of the estimated entropies measured for the degree of concentrations using vector distributions.     

基于双隐含层BP算法的激光主动成像识别系统

在传统激光主动成像系统的基础上,结合目标识别技术搭建了一个激光主动成像识别系统实验平台,用于研究激光主动成像后的目标识别。介绍了实验平台的工作原理,基于Hu矩特征的双隐含层BP神经网络算法以及实验处理流程和实验结果。特征量由7个不变Hu矩构成,通过240张原始目标样本库对由136个权值系数构成的双隐含层BP神经网络算法进行了训练。利用训练好的双隐含层BP算法对黑夜条件下远处的运动目标--43式冲锋模具枪进行了实验研究,成功获得了清晰的红外激光主动成像效果。实验显示对450 m处2 740帧和550 m处2 420帧激光主动成像图像的统计识别率达到了68.87%和72.11%,其中旋转变换下的统计识别率可达80.05%和84%,好于仿射变换的识别效果。     

Design and use of a computer controlled confocal microscope for biological applications

In the confocal scanning light microscope where the object is scanned mechanically through a finely focused laser spot, a fundamentally higher resolution can be achieved when a point detector is employed. Such a microscope possesses in addition to a high dynamic range excellent sectioning capabilities, and is because of this very suitable for “3-Dimensional imaging”, especially when operated in fluorescence. An instrument under computer control and with extensive image processing capabilities is described. Various biological applications are given including computer generated stereo images of biological structures with submicron resolution.     

Large-area, high-resolution image analysis of composite materials

An image analysis system based on a small network of parallel processors hosted within a PC has been developed to measure the orientations of glass and carbon fibres in a polymer matrix from a polished section using optical microscopy. A computer-controlled XY translation stage is used to scan a large sample area (mm × mm), at a high magnification to minimize measurement errors. The distance that the stage moves between adjacent image frames is accurately measured allowing partial images at the edges of image frames to be reconstructed and so produce a continuous area. By removing a small amount of material from the sample by polishing and rescanning the same sample area, data from a number of serial sections can be obtained. Pattern matching is then used to correlate the fibre images from each section, allowing full three-dimensional orientation data to be extracted, overcoming the limitations of measurements from a single, two-dimensional section plane.     

Flat field correction for high‐throughput imaging of fluorescent samples

Vignetting of microscopic images impacts both the visual impression of the images and any image analysis applied to it. Especially in high‐throughput screening high demands are made on an automated image analysis. In our work we focused on fluorescent samples and found that two profiles (background and foreground) for each imaging channel need to be estimated to achieve a sufficiently flat image after correction. We have developed a method which runs completely unsupervised on a wide range of assays. By adding a reliable internal quality control we mitigate the risk of introducing artefacts into sample images through correction. The method requires hundreds of images for the foreground profile, thus limiting its application to high‐throughput screening where this requirement is fulfilled in routine operation.     

Sequential processing of quantitative phase images for the study of cell behaviour in real‐time digital holographic microscopy

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time‐lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long‐term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time‐lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least‐squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real‐time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off‐axis holographic microscope.     

多尺度光斑中心的快速检测

分析了光斑图像成像特点和理想光斑灰度分布模型,针对含有多个不同尺度光斑的图像,提出了一种可以在复杂环境下一次性快速检测出多个光斑中心的方法。该方法基于高斯模糊后光斑中心不变的性质,先对含有大量光斑的图像进行快速多级高斯模糊,构建其高斯尺度空间;然后,使用加速的非极大值抑制方法在尺度空间内寻找多个尺度的局部极值,初步确定各光斑中心的像素级坐标;最后,联合这些坐标的邻域像素,拟合局部曲面,得到光斑中心的亚像素级精确位置。利用仿真实验和实物实验验证了提出方法的有效性。结果表明:该算法对640pixel×480pixel图像,处理时间仅需50ms,每千个光斑的平均检测时间为23ms,在复杂环境下正确率可达89%。此外,该方法对弱光斑较敏感,适合快速处理含有大量不同尺度光斑的图像,并能够有效减少光斑的错检和漏检。由于检测速度快,自适应性强,在实际应用中取得了良好的检测效果。     

高性能数字显微图像系统的研制

数字显微图像技术是传统目视光学显微图像观察手段的现代化、信息化发展。这里介绍了一种高性能数字显微图像系统，对研制过程中所涉及的图像光电变换、图像时－空分辨率改善、分析专用软件设计等关键性技术问题作了阐述，并给出了若干典型应用实例。     

在线图像可视铁谱成像系统的像面照度均匀性

为了定量评价在线图像可视铁谱(OLVF)成像系统的像面照度均匀性,建立了一种像面照度模型。以像方参数及放大倍率表征物方视场,将物方视场区域离散化,采用朗伯余弦理论建立入瞳模式的像面照度模型,实现了像面照度的计算与均匀性评价。利用Matlab进行了物面照度仿真分析,确定了OLVF成像系统环形阵列光源的发光二极管(LED)数量,基于像面照度分析确定了最佳成像焦距和放大倍率。计算了油腔通油情况下成像系统中的光能量损耗以及磨粒沉积面的照度分布,建立了油液吸光系数与CCD像面轴上像点照度的关系。结果显示:LED发光强度已知时,仿真计算的像面不均匀度约为5.60%,实际测试的像面不均匀度为8%~9%,满足不均匀度≤10%的要求。开展了磨粒铁谱图像采集实验,结果表明:图像中磨粒清晰可辨,便于图像分割与视觉特征提取。提出的模型可定量描述OLVF成像系统的像面照度,可作为优化系统结构,提高系统成像性能的依据。     

全视场在线图像可视铁谱磨粒显微成像特性分析

为了评估全视场在线图像可视铁谱磨粒显微成像特性,提出了一种反射光显微成像模型。首先,基于朗伯余弦与小角度散射理论建立了全视场(OLVF)的反射光辐照度模型,实现了磨粒显微成像清晰度定量评价。然后,仿真计算磨粒显微成像的反差透视比,确定了最优光学倍率和适用于全视场OLVF探测的油液衰减系数范围,明确了光学参数对磨粒显微成像质量的影响规律。结果显示：光学倍率为2.0×且油液衰减系数≤2.0条件下,磨粒沉积于物方视场的主光轴附近,全视场OLVF可获得最佳磨粒显微成像清晰度。最后,开展了磨粒显微成像实验测试,结果表明：全视场OLVF能够从油液衰减系数小于2.0的在用液压油和齿轮油中获取反射光谱片图像,并提取磨粒视觉信息进行磨损在线监测。     

Multiparametric high-resolution imaging of barley embryos by multiphoton microscopy and magnetic resonance micro-imaging

Nonlinear optical microscopy and magnetic resonance imaging (MRI) address different properties of the sample and operate on different geometrical scales. MRI maps density and mobility of molecules tracking specific molecular signatures. Multiphoton imaging profits from the nonlinear absorption of light in the focus of a femtosecond laser source stimulating the autofluorescence of biomolecules. As this effect relies on a high light intensity, the accessible field of view is limited, but the resolution is very high (a few hundred nanometers). Here, we aim to link the different accessible scales and properties addressed in the different techniques to obtain a synoptic view. As model specimen we studied embryos of barley. Multiphoton stimulated autofluorescence images and images of second harmonic generation are achieved even down to low magnification (10x), low numerical aperture (N.A. 0.25) conditions. The overview images allowed morphological assignments and fluorescence lifetime imaging provides further information to identify accumulation of endogenous fluorophores. The second, complementary contribution from high-resolution MR images provides a 3D model and shows the embedding of the embryo in the grain. Images of the proton density were acquired using a standard 3D spin-echo imaging pulse sequence. Details directly comparable to the low magnification optical data are visible. Eventually, passing from the MR images of the whole grain via low magnification to high resolution autofluorescence data bridges the scale barrier, and might provide the possibility to trace transport and accumulation of, e.g., nutrients from large structure of the plant to the (sub-) cellular level.     

异步Binning高帧频CCD相机的拖尾校正

一种基于异步时序驱动和Binning技术的高帧频成像技术在帧转移CCD相机上得到成功应用。为了抑制该CCD相机成像时产生的拖尾效应,基于该相机的高帧频连续成像原理,理论上分析了图像获取过程中拖尾产生的原因,给出了一套完整的消除拖尾的数值解析算法。该算法充分考虑了图像亮度变化对拖尾形成产生的影响,并对此进行了修正,已在异步Binning CCD相机上进行了实验验证。利用相机以40 000帧每秒的帧频获取4幅连续图像进行实验,实验结果表明,该算法可有效去除每幅图像的拖尾噪声,而且消除拖尾噪声后的图像区域的灰度方差得到明显降低,异步Binning CCD相机的成像质量得以提高。上述结果表明算法适用于高帧频相机的连续成像。     

The imaging mechanism of single-walled carbon nanotubes on Si/SiO₂ wafer in scanning electron microscopy

Scanning electron microscopy imaging of both suspended single‐walled carbon nanotubes (SWNTs) and contacted SWNTs with Si/SiO₂ substrate has been studied in this paper. The voltage contrast has been investigated by supplying external electric field around the samples. The results show that the image contrast of SWNTs attributes to both voltage contrast from the area surrounding SWNTs (tens of nanometres in both sides of the SWNTs) and electron beam induced emission from SWNTs themselves under low primary beam energy. Under high primary beam energy, however, EBIE dominates the image contrast due to the fact that the voltage contrast caused by implanted charges of the SiO₂ layer is weakened. Imaging under the primary beam energy lower than 1 keV offers widened diameter of SWNTs, which promises that the SWNTs are observable at very low magnification (lower than 100×). At a larger magnification, however, imaging under the primary beam energy higher than 10 keV can display more realistic images of the SWNTs. In addition, an appropriate external electric field can improve the images.     

Seamless stitching of tile scan microscope images

For diagnostic purposes, optical imaging techniques need to obtain high‐resolution images of extended biological specimens in reasonable time. The field of view of an objective lens, however, is often smaller than the sample size. To image the whole sample, laser scanning microscopes acquire tile scans that are stitched into larger mosaics. The appearance of such image mosaics is affected by visible edge artefacts that arise from various optical aberrations which manifest in grey level jumps across tile boundaries. In this contribution, a technique for stitching tiles into a seamless mosaic is presented. The stitching algorithm operates by equilibrating neighbouring edges and forcing the brightness at corners to a common value. The corrected image mosaics appear to be free from stitching artefacts and are, therefore, suited for further image analysis procedures. The contribution presents a novel method to seamlessly stitch tiles captured by a laser scanning microscope into a large mosaic. The motivation for the work is the failure of currently existing methods for stitching nonlinear, multimodal images captured by our microscopic setups. Our method eliminates the visible edge artefacts that appear between neighbouring tiles by taking into account the overall illumination differences among tiles in such mosaics. The algorithm first corrects the nonuniform brightness that exists within each of the tiles. It then compensates for grey level differences across tile boundaries by equilibrating neighbouring edges and forcing the brightness at the corners to a common value. After these artefacts have been removed further image analysis procedures can be applied on the microscopic images. Even though the solution presented here is tailored for the aforementioned specific case, it could be easily adapted to other contexts where image tiles are assembled into mosaics such as in astronomical or satellite photos.     

用于线纹显微图像的边缘检测算法

为了在简化计算的同时达到较高的定位精度,提出一种轴向邻域和差边缘检测算法,用于低信噪比、缓慢过渡的微结构显微图像的边缘检测。首先,结合显微图像采集系统的配置,分析了线纹显微图像边缘灰度轮廓特征和基于导数的边缘检测算法的不足。然后,基于方向信息测度,定义了轴向邻域和差运算,依据矩不变理论推导出轴向邻域和差边缘检测算法。实验结果表明:轴向邻域和差边缘检测算法能够适应不同分辨率的显微图像,具有较强的抗噪能力和较高的定位精度,边缘检测效果优于基于导数的算法。该算法用于显微图像时,其边缘坐标定位方差为0.57pixel,微米级线条宽度的测量结果与扫描电子显微镜的测量结果(1.35μm)相差0.17μm,基本满足了测量精度的要求。     

克服动态问题影响的相机响应函数标定

利用图像之间的亮度映射函数而不是图像本身各像素点信息进行相机响应函数的标定,避免了成像系统的晃动或者拍摄场景的动态问题带来的图像配准之间的误差给后续标定算法带来的影响。采用直方图规则化的方法分析不同曝光量图像之间的各颜色通道不同亮度级的统计特征,获得了不同图像对之间的亮度映射函数。然后,利用图像对之间的亮度映射函数结合各帧图像的直方图建立求解相机响应函数的超定方程模型。最后,利用最小二乘法解算模型获得相机响应函数。验证实验表明,本文相机响应函数标定算法具有克服相机的动态问题的能力;不同输入帧数图像的分组实验显示,最小输入4帧图像能够获得较为理想的响应函数标定结果。