Curvature measurement with reflected-light microscopy and its application to fly facet lenses

Two methods for microscopically measuring the curvature of strongly curved surfaces are compared: one using a Michelson interferometer-type microscope and one using a novel reflectometrical method implemented in an epi-illumination microscope. The curvature values obtained with the two methods were very similar, but the latter proved to be by far the simplest. Curvature measurements on the front surface of the facet lenses of various dipteran flies revealed that facet lens diameter and radius of curvature are linearly related over a wide range of facet lens sizes.     

Light exposure and cell viability in fluorescence microscopy

Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm^?2 and about 300 J cm^?2 for native cells (corresponding to about 250?3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi‐ or total internal reflection illumination) and decreased to values between 50 J cm^?2 and less than 1 J cm^?2 upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high‐resolution wide field or laser scanning microscopy of single cells, typically 10?20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super‐resolution microscopy based on single molecule detection.     

Mirrored pyramidal wells for simultaneous multiple vantage point microscopy

We report a novel method for obtaining simultaneous images from multiple vantage points of a microscopic specimen using size‐matched microscopic mirrors created from anisotropically etched silicon. The resulting pyramidal wells enable bright‐field and fluorescent side‐view images, and when combined with z‐sectioning, provide additional information for 3D reconstructions of the specimen. We have demonstrated the 3D localization and tracking over time of the centrosome of a live Dictyostelium discoideum. The simultaneous acquisition of images from multiple perspectives also provides a five‐fold increase in the theoretical collection efficiency of emitted photons, a property which may be useful for low‐light imaging modalities such as bioluminescence, or low abundance surface‐marker labelling.     

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 μm Araldite-embeddedhistologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 μm Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and “expansion chambers” may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.     

基于单次成像的三维形貌拼接技术

针对大型物体的三维形貌测量,研究基于辅助靶标的三维形貌拼接技术,并在此基础上提出单摄像机单次成像求解转换矩阵的方法.该方法通过单摄像机单次成像,结合控制点的边长约束,求解出全局坐标系同局部测量坐标系的转换关系,进而将局部测量数据统一到全局坐标系下,实现拼接.此方法避免采用单摄像机多摄站测量时带来的繁琐操作以及产生的相关问题,大大提高拼接的速度,并且可以保证较高的精度.     

基于OpenGL的双目摄像机的实时3D显示

文章阐述了3D显示的基本原理以及几种主流的3D显示实现方法,并介绍了在Visual C++6.0环境下,如何应用开放图形库——OpenGL实现双目立体视觉摄像机的立体显示。     

Defocusing microscopy

Transparent objects (phase objects) are not visible in a standard brightfield optical microscope. In order to see such objects the most used technique is phase-contrast microscopy. In phase-contrast microscopy the contrast observed is proportional to the optical path difference introduced by the object. If the index of refraction is uniform, phase-contrast microscopy then yields a measure of the thickness profile of phase objects. We show that by slightly defocusing an optical microscope operating in brightfield, phase objects become visible. We modeled such an effect and show that the image contrast of a phase object is proportional to the amount of defocusing and proportional to the two-dimensional Laplacian of the optical path difference introduced by the object. For uniform index of refraction, defocusing microscopy then yields a measure of the curvature profile of phase objects. We extended our previous model for thin objects to thick objects. To check our theoretical model, we use as phase objects polystyrene spherical caps and compare their curvature radii obtained by defocusing microscopy (DM) to those obtained with atomic force microscopy (AFM). We also show that for thick curved phase objects one can reconstruct their thickness profiles from DM images. We illustrate the utility of defocusing microscopy in biological systems to study cell motility. In particular, we visualize and quantitatively measure real-time cytoskeleton curvature fluctuations of macrophages (a cell of the innate immune system). The study of such fluctuations might be important for a better understanding of the engulfment process of pathogens during phagocytosis.     

Super-resolution microscopy using normal flow decoding and geometric constraints

Prior knowledge about the observed scene provides the key to restoration of frequencies beyond the bandpass of an imaging system (super-resolution). In conjunction with microscopy two super-resolution mechanisms have been mainly reported: analytic continuation of the frequency spectrum, and constrained image deconvolution. This paper describes an alternative approach to super-resolution. Prior knowledge is imposed through geometric and dynamic models of the scene. We illustrate our concept based on the stereo reconstruction of a micropipette moving in close proximity to a stationary target object. Information about the shape and the movement of the pipette is incorporated into the reconstruction algorithm. The algorithm was tested in a microrobot environment, where the pipette tip was tracked at sub-Rayleigh distances to the target. Based on the tracking results, a machine vision module controlled the manipulation of microscopic objects, e.g. latex beads or diamond mono-crystals. In the theoretical part of this paper we prove that knowledge of the form 'the pipette has moved between two consecutive frames of the movie' must result in a twofold increase in resolution. We used the normal flow of an image sequence to decode positional measures from motion evidence. In practice, super-resolution factors between 3 and 5 were obtained. The additional gain originates from the geometric constraints that were imposed upon the stereo reconstruction of the pipette axis.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

数字全息三维显示关键技术与系统综述

三维全息显示能够表现出与真实物体一样的深度和视差,是一种理想的三维显示方法。但是,三维物体计算全息图计算复杂且计算量巨大,因此,如何快速生成三维物体计算全息图是数字三维全息动态显示中的关键问题之一。本文首先论述了数字全息三维显示的关键技术,包括物点散射法、体视全息法、层析法等三种三维物体计算全息图实现方法,一种RGB分离的真彩色全息显示实现方法和若干提高全息再现像质的方法;然后对几种最新典型的数字三维全息显示系统进行了技术分析;最后总结了数字全息三维显示领域的发展动态,指出三维全息显示技术会朝着实时、动态、更大尺寸、更高分辨率方向发展。     

Reconstruction of the complex reflectance function in acoustic microscopy

We describe the theory and practical implementation of an iterative computer algorithm (an extension of the Gerchberg Saxton algorithm, originally developed for electron microscopy) for enabling an estimate of the complex reflectance function of a material to be reconstructed from measured values of only the magnitude of the response of an acoustic microscope (i.e. without the phase of the transducer voltage). Results are presented for eight materials measured with a spherical lens (at 320 MHz) and five with a cylindrical line-focus lens (at 210 and 228 MHz).     

Application of regularized Richardson-Lucy algorithm for deconvolution of confocal microscopy images

Although confocal microscopes have considerably smaller contribution of out-of-focus light than widefield microscopes, the confocal images can still be enhanced mathematically if the optical and data acquisition effects are accounted for. For that, several deconvolution algorithms have been proposed. As a practical solution, maximum-likelihood algorithms with regularization have been used. However, the choice of regularization parameters is often unknown although it has considerable effect on the result of deconvolution process. The aims of this work were: to find good estimates of deconvolution parameters; and to develop an open source software package that would allow testing different deconvolution algorithms and that would be easy to use in practice. Here, Richardson-Lucy algorithm has been implemented together with the total variation regularization in an open source software package IOCBio Microscope. The influence of total variation regularization on deconvolution process is determined by one parameter. We derived a formula to estimate this regularization parameter automatically from the images as the algorithm progresses. To assess the effectiveness of this algorithm, synthetic images were composed on the basis of confocal images of rat cardiomyocytes. From the analysis of deconvolved results, we have determined under which conditions our estimation of total variation regularization parameter gives good results. The estimated total variation regularization parameter can be monitored during deconvolution process and used as a stopping criterion. An inverse relation between the optimal regularization parameter and the peak signal-to-noise ratio of an image is shown. Finally, we demonstrate the use of the developed software by deconvolving images of rat cardiomyocytes with stained mitochondria and sarcolemma obtained by confocal and widefield microscopes.     

基于单目视觉的自由曲面三维坐标测量方法

本文将单摄像机与三坐标测量机结合设计了一种自由曲面的三维坐标测量方法.自由曲面的表面经坐标测量机带动摄像机序列采集图像后形成图像体积,然后使用调焦评价函数在图像体积内寻优判断,找到自由曲面的正焦图像表面.根据物像关系式和正焦图像表面即可推得自由曲面点的三维坐标.经实验比较,本系统测量结果与接触式坐标测量机测量曲面点Z坐标极限偏差13 μm.     

Space-multiplexed multifocal nonlinear microscopy

Standard forms of nonlinear microscopy rely on single beam scanning, but the usually weaker signal and the need to image in real-time call for parallelization of the image formation. Since the nonlinear susceptibilities necessitate a comparatively large illumination power, with current laser systems the brightness or field of view of any parallelized nonlinear microscope is limited by the brightness of the laser. For example, by producing an array of high aperture foci, multifocal multiphoton microscopy (MMM) provides real-time, light-efficient three-dimensional fluorescence imaging at high-resolution. The available power limits the degree of parallelization and hence codetermines the field of view. As the utilization of all the laser power is imperative, the focal intensity can be adjusted only through altering the number of foci. This compromises to some extent the flexibility to adjust the focal intensity to benign and effective levels. Here we introduce space-multiplexing (SMX) as a novel option in parallelized nonlinear microscopy, which enables an improved exploitation of the total laser power and facilitates changing the intensity levels in selected regions, without attenuating the total laser power. The basic idea of SMX is to overlap arrays of slightly offset coherent focal fields whose interference modulates the intensity across the sample. For a given degree of parallelization and power, SMX increases the two- and three-photon excited signal of parallelized nonlinear microscopy by a factor of up to 1.5 and 2.5, respectively. To some extent, sensitive regions may be spared out, whereas in regions with weaker nonlinear susceptibilities the intensity is increased. SMX is relevant to all modes of nonlinear microscopy, including parallelized second- and third-harmonic imaging, coherent anti-Stokes Raman scattering, and widefield multiphoton excitation.     

基于虚拟仪器的三维视觉检测系统设计

把计算机视觉领域中的明暗恢复形状(Shape From Shading,简称SFS)技术应用于工业产品的三维视觉检测,利用单幅产品图像的明暗灰度变化来得到物体表面形状信息,并采用全面像素不确定度进行检测分析。该检测系统采用Lab VIEW及其视觉开发工具来实现,可广泛应用于工业自动在线三维视觉形貌检测和质量控制。     

会展业虚拟展示系统的设计与实现

针对文物网上虚拟展示的需求,设计了一种基于C/S的会展业虚拟展示系统。本文主要介绍了VRML的工作原理,会展业虚拟会展系统的总体设计以及客户端与服务器端的设计。系统提供设计展馆的功能,用户可根据用户个人爱好选择不同的展示主题。此外,系统还提供用户参观路径的自由选择、地图导游、感兴趣文物浏览等功能,提供文物多媒体信息集成展示。最终以良渚文化博物馆展品为对象,进行了应用示范,达到了预期的效果。     

大尺度钢板的三维测量和拼接

提出了基于投影的三维测量拼接方法,用于测量大尺度钢板表面的三维形状。首先,利用光学扫描仪、背景投影仪两种装置实现大尺度钢板的三维测量。其中,三维光学扫描仪负责测量大尺度钢板不同部分的三维数据,背景投影仪用于向被测钢板投射背景纹理;然后,利用基于随机抽样一致性算法（RANSAC）的拼接算法,将不同时刻测量的局部三维数据进行拼接,得到完整的钢板三维数据;最后,提出了一种拼接误差的评价方式来检验拼接精度。实验结果表明：所提方法的单次拼接精度为0.5mm左右;测量一个7.5m长的钢板,其累计拼接误差为2mm左右。得到的结果基本满足船舶外板加工的精度要求,具有较高的实用价值。     

网条结构光三维视觉检测标定方法研究

本文在广义坐标系中采用投影透视变换理论建立了网条结构光三维视觉检测模型。     

3‐D PSF fitting for fluorescence microscopy: implementation and localization application

Localization microscopy relies on computationally efficient Gaussian approximations of the point spread function for the calculation of fluorophore positions. Theoretical predictions show that under specific experimental conditions, localization accuracy is significantly improved when the localization is performed using a more realistic model. Here, we show how this can be achieved by considering three‐dimensional (3‐D) point spread function models for the wide field microscope. We introduce a least‐squares point spread function fitting framework that utilizes the Gibson and Lanni model and propose a computationally efficient way for evaluating its derivative functions. We demonstrate the usefulness of the proposed approach with algorithms for particle localization and defocus estimation, both implemented as plugins for ImageJ.