激光共焦高速扫描显微成像的高帧速重构算法

针对激光共焦扫描显微镜的往复式逐行扫描成像方式带来的帧图像数据分割难的问题,在分析系统扫描方式、振镜的实际运动方式与理论运动方式差异的基础上,利用相邻两帧图像相似性大的特点,提出了一套完整的高帧速重构算法。该算法通过连续帧特征区域差分的方式实现了一维信号序列的自适应分割,即实现了对一维信号序列进行动态排列及分割成二维阵列图像数据,从而重构出多帧高精度图像。实验表明,该算法的成像误差低于1.6%,适用于成像速度高达300帧/s的激光共焦扫描显微成像。     

高速扫描激光共聚焦显微内窥镜图像校正

使用往复式逐行扫描的方式可以提高激光共聚焦显微内窥镜的成像速度和数据利用率,但这种扫描方式也会带来图像畸变和错位问题,从而影响系统成像质量。受扫描畸变影响,图像错位程度不一致,后期处理难以得到理想的校正效果。本文基于共振振镜的运动规律,推导了均匀空间采样过程中的采样时间函数,通过非等时采样方法校正了振镜速度变化带来的横向畸变。利用互相关法评价图像错位程度,采用遗传算法获得最优的采样开始时刻,实现了图像错位的校正。最终通过调整采样开始时刻和时间间隔在数据采集环节校正了图像畸变和错位。为了验证图像畸变校正和错位校正效果,本文搭建了基于往复式逐行扫描方式的激光共聚焦显微内窥成像系统。实验结果表明,该方法能够有效地校正图像畸变和错位,图像的横向分辨率。与现有方法相比,本文方法将图像的局部分辨率由10 pixel提高到6 pixel。     

基于连续扫描方式的激光共焦扫描显微镜的研制

研发了一套基于连续扫描的激光共焦扫描显微镜(laser confocal scanning microscopy,LCSM)系统。该系统采用工作台连续运动方式实现扫描,提出了利用单次采集的数据滤除随机噪声的方法,避免了多帧取平均对成像速度造成的影响。实现连续扫描的关键在于解决工作台运动与数据采集的同步问题,利用采集卡有限采集模式,合理匹配工作台参数和采集参数,成功解决了这一问题。详细介绍了影响分辨率的因素,通过合理选取探测器针孔直径,取样间隔,确保了实现高分辨率的要求。系统利用Visual C#开发的控制平台,成功地对生物细胞进行了扫描成像。实验结果表明:基于连续扫描的LCSM具有较高的分辨率,生成的显微图像没有任何畸变,并且成像速度有了大幅度提高。     

激光共焦扫描显微镜简介

激光共焦扫描显微镜是显微光学技术发展史上的新里程碑,它集激光技术、光学技术、电子计算机技术,作为现代化的显微成像系统倍受广大科研工作者的关注,并在生命科学、材料科学等领域中具有广泛的应用。本文就激光共焦扫描显微镜的主要技术特点作综合性介绍。     

基于线扫描方式的激光共焦显微镜的研究

激光共焦扫描显微镜(LCSM)是一种新型光学显微镜,它能够对活体组织的深度结构进行清晰的二维或三维成像.由于传统共焦扫描显微镜采用单点扫描方式,成像速度慢,无法满足活体组织实时成像的要求.介绍一种以线扫描方式代替点扫描方式的激光共焦扫描显微镜.线扫描方式不但简化了扫描机构的设计,而且提高了扫描速度,使活体组织的实时成像成为可能.     

国外简讯

<正> MRC-600激光共焦显微成像系统英国医学研究协会和伯乐(Bio-Rad)显微部门联合研制的MRC-600激光共焦显微成像系统,于最近由伯乐公司投放市场。该仪器能穿透地观察到样品的立体分层结构(如DNA单股或双股螺旋结构,细施癌变过程的讯号转导过程等);对于荧光免疫的实验样品,显像反差效果甚佳,从而有利于开展分     

光纤共聚焦显微内窥镜活体内实时成像系统的设计和研究

介绍了一种光纤共聚焦显微内窥镜实时活体内成像系统。该系统采用高速扫描振镜、超细成像光纤束、大尺度几何形变理论图像拼接技术,并结合自主研发的综合软件,使该系统具有实时检测、探头物理尺寸小、图像分辨力高、用户界面友好、操作方便等多方面优势。实验表明:该系统可为医生提供丰富的组织学和病理学影像信息,提高诊断准确率。该系统为临床医学提供了一种能在活体内进行实时细胞尺寸检测的医疗仪器,是癌症早期诊断的重要工具。     

共振扫描显微成像中的图像畸变校正

设计了一种校正算法用于校正双光子荧光显微镜等高速扫描成像系统中共振振镜扫描导致的图像畸变。首先对共振振镜的扫描运动建立模型,推导出非线性扫描的运动公式,进而得到图像畸变公式;然后对一块朗奇光栅样品扫描成像,设计了多峰高斯拟合算法得到光栅所有条纹的宽度变化并通过最小二乘法将条纹宽度数据拟合成一条畸变曲线;最后利用畸变曲线对图像进行校正。结果表明:采用提出的校正算法可使系统最大畸变减小到传统正弦校正方法的1/3,相对畸变减小到1/5,校正效果比传统的正弦校正法提高了2倍。由于提出的曲线拟合校正算法不用增加额外的光路,且不需要切割边缘图像,故显示了极好的图像使用效率和校正效果。     

一种大视野超分辨结构光照明显微成像方法

近年来结构光照明显微术(SIM)在技术发展和应用方面都受到了广泛关注。然而,传统采用空间光调制器(SLM)进行条纹投影的SIM成像视野有限。为满足生物医学研究中高通量显微成像的需求,本文基于SIM开发一种基于激光干涉的大视野超分辨荧光显微成像系统。将用于投射条纹图案的2D光栅和用于控制条纹方向相移的SLM相结合,突破数字投影设备对条纹数量和精细度的限制。开发一种空域重建超分辨图像的算法来提高成像速度。最后,搭建1套结构光照明荧光显微成像系统样机,在20×放大倍数和物镜NA 0.75的条件下,验证了本文方法的有效性,在分辨率提升至1.8倍的同时视野可达1 380μm×1 035μm,所述空域超分重建算法能够大幅降低计算耗时。     

动态聚焦激光振镜扫描系统的误差分析及图形校正算法

动态聚焦激光振镜扫描系统广泛应用于大幅面激光扫描领域,在实现振镜高速扫描的同时,必须要保证扫描图形的精度。本文从动态聚焦激光振镜扫描系统的原理入手,分析了动态聚焦激光振镜扫描系统图形误差产生的原因,并给出了扫描图形的误差校正模型和精确校正算法,并在华中科技大学快速成型中心的粉末烧结快速成型设备上进行了试验验证。     

Laser scanning phase modulation microscope

We describe the concept and first implementation of an innovative new instrument for quantitative light microscopy. Currently, it provides selective imaging of optical path differences due to birefringence; with further development, it is also possible to selectively image several optical properties, including refractive path differences, optical rotation, and linear and circular dichroism, all with diffraction-limited resolution. An image consists of a 512×512 element array, with each pixel displaying one of 256 grey levels, linearly proportional to the specific optical property being observed. Additionally, conventional brightfield and polarized light microscopy are available, with the accompanying advantages of laser scanning and digital image processing. The microscope consists of three subsystems, representing three distinct technologies. The laser scanning subsystem moves a focused, microspot across the specimen; the output of a photodetector is an electric signal corresponding to a scanned image. The image display subsystem digitizes this signal and displays it as an image on a video monitor. When used in conjunction with a phase modulation feedback loop, the image formed is of the specimen's birefringent retardation or other selected optical property. The digitized images are also available for computer enhancement.     

Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope

High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.     

A new confocal scanning beam laser MACROscope using a telecentric,f-theta laser scan lens

A new confocal scanning beam system (MACROscope) that images very large-area specimens is described. The MACROscope uses a telecentric, f-theta laser scan lens as an objective lens to image specimens as large as 7·5 cm × 7·5 cm in 5 s. The lateral resolution of the MACROscope is 5 μm and the axial resolution is 200 μm. When combined with a confocal microscope, a new hybrid imaging system is produced that uses the advantages of small-area, high-speed, high-resolution microscopy (0·2 μm lateral and 0·4 μm axial resolution) with the large-area, high-speed, good-resolution imaging of the MACROscope. The advantages of the microscope/MACROscope are illustrated in applications which include reflected-light confocal images of biological specimens, DNA sequencing gels, latent fingerprints and photoluminescence imaging of porous silicon.     

长波红外相机在轨扫描成像畸变消除控制策略

为消除长波红外相机在轨扫描成像的地面畸变,研究了变焦扫描成像畸变消除系统控制技术。针对变焦扫描控制的多电机位置同步要求,提出一种高精度多电机位置同步算法。依据设计的变焦扫描成像控制原理,设定每个扫描时刻点变倍组电机和补偿组电机的同步位置。变焦控制系统实现与扫描控制系统的同步计时,根据当前位置和下一时刻的同步位置,每隔0.01s计算出电机的运行速度,实时对电机的速度进行控制。实验结果表明,变倍组和补偿组电机的位置同步偏差分别在±0.003mm、±0.002mm以内;沿轨方向地面分辨率的最大偏差不超过±0.047m。长波红外相机在连续变焦扫描控制过程中成像清晰,达到消除畸变的效果,满足变焦扫描成像的要求。     

A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 μm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.     

Confocal imaging with bilateral scanning and array detectors

A novel arrangement for confocal microscopy is presented, in which the key elements are the use of an array detector such as a CCD for confocal image collection and the use of one double-sided scanning mirror element for bilaterally scanning the object and collecting the data on the CCD. The resulting arrangement is shown to be capable of confocal imaging with high photon efficiency under adjustable conditions of confocality and varying image acquisition rates, i.e. from slow speed up to real-time imaging. Either laser or conventional light sources may be utilized. In addition to CCD registration, direct observation by eye of the confocal image in fluorescence is also possible.     

Digital beam control for fast differential voltage contrast

A new method for voltage contrast enhancement by image subtraction is presented. It is based on a modification of a commercial column automation system that digitizes beam positions and intensity signals in a scanning electron microscope (SEM). The new modulus performs via hardware all the operations of bias control and signal differentiation at each image point, resulting in a minimum acquisition time of 4 μs per point, that is less than 0.3 seconds to obtain a 256 × 256 pixels image with 8 bit gray resolution. The digital handling of intensity data, gives high precision differential images and the control of beam position prevents spatial differentiation effects, enabling noise reduction by time integration of intensity data in each point.     

光机二维扫描技术在激光共聚焦生物芯片扫描仪中的应用

介绍了应用于激光共聚焦生物芯片扫描仪中的光机二维扫描技术 ,即用振镜和f- θ扫描物镜构成其中一维的光扫描系统 ,用步进电机驱动扫描工作台移动构成另一维机械扫描系统 ,并在此基础上分析研究了光机二维扫描控制系统的设计。为快速、高精度激光共聚焦生物芯片扫描仪的研制作了新的有益尝试 ,并取得了初步的实验结果