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。     

预测扫描提前跳转的电子稳像方法

提出一种基于传感器电子稳像技术的扫描稳像方法———预测扫描提前跳转法,用以处理电子稳像过程中摄像系统的扫描运动问题。首先,利用传感器敏感摄像系统运动,实现对图像序列帧间偏移量的估计,并根据估计偏移量预测图像序列是否存在扫描运动;然后,对存在扫描运动的图像将参考帧提前跳转到扫描后位置,再运用稳像算法进行处理。该方法通过提前跳转参考帧将扫描模式下的稳像过程简化为不存在扫描运动的稳像过程,简化了扫描稳像的数学模型,降低了算法复杂度,同时通过跳转步长的设置和缓存参考帧技术进一步提高了算法的准确度和可靠性。实验结果表明,该方法可在输出稳定图像的同时正确显示视频序列的扫描运动。     

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.     

激光共焦显微光束的偏转扫描

为了提高激光共聚焦系统的扫描速度,本文提出一种逐场扫描的场同步扫描方法。构建了激光共焦显微系统,将美国THORLABS公司的GVS002型二维检流计振镜应用于该系统,根据光学系统参数以及扫描范围要求计算振镜的整场扫描波形。借助NI公司的PCIe6353多功能数据采集卡,输出行同步的扫描波形,同时,对共焦显微系统共焦位置上针孔处的光强信号进行采集,先后扫描一幅256×256和512×512的图像,记录扫描图像和成像时间;然后,在相同的硬件结构下,以场同步的方式输出扫描波形,记录扫描图像和成像时间。实验结果表明:场同步方式扫描256×256图像的速度可提高10倍,扫描512×512图像的速度可提高5倍,且满足共焦显微成像的清晰、抗干扰能力强等要求。与行同步扫描方法相比,场同步扫描方法可以消除行与行之间转换的停留时间,在不改变硬件的情况下大幅提高扫描速度。     

分焦平面红外偏振微扫描图像超分辨重建

分焦平面偏振探测系统受其探测器结构的影响,成像分辨率低于探测器实际分辨率,本文在不改变光学系统结构下使用微扫描获取亚像元微位移帧序列,提出一种改进的凸集投影(Projection On Convex Sets, POCS)算法用于提升偏振成像系统的成像分辨率。该算法首先对获取到的偏振微扫描图像序列进行检偏角分离,将同组检偏角图像序列作为输入,其次进行位移匹配与凸集投影迭代初步重建高分辨率图像,然后将图像分组进行滑动窗口非邻域聚类,利用主成分分析将聚类后的图像进行降维,最后将每一维信息视为时间采样函数,在小波域进行软阈值降噪。实验表明,本算法可以有效的提高传统POCS算法的抗噪性能,提高分焦平面偏振探测系统的成像分辨率,和同类算法相比结构相似性系数提升0.02,峰值信噪比提升约1 dB,并且拥有更高的噪声鲁棒性。     

航空光电成像电子稳像技术

介绍了一种电子稳像技术,解决了航空平台光电成像系统视频图像帧内运动模糊和帧间不稳定问题。通过建立运动模糊的数学模型,构建二维运动模糊点扩散函数,采用维纳滤波方法,消除图像帧内运动模糊;通过灰度投影算法,检测序列图像当前帧和参考帧之间的运动矢量,采用图像补偿方法,实现序列图像稳定输出。实验结果表明,本文方法能提高信噪比,实现1 pixel的稳定精度,有效改善图像质量,输出清晰稳定的视频图像, 具有精确、快速、实用的特点。     

基于图像融合的运动目标检测与跟踪方法研究

从运动背景中检测与跟踪运动目标是计算机视觉研究领域的热点,根据帧差法的基本原理,提出了一种针对复杂背景的运动目标检测方法.首先通过设定阈值滤除序列图像中的噪声,然后对三帧算法进行改进,即利用序列中多帧图像融合运动信息,并确定参考区域,通过对原图像进行回扫描,最终提取出完整的运动目标轮廓.最后采用一种运动物体跟踪算法,实现了运动物体和静止物体的识别,克服了以往算法中的误检和空洞问题,实验结果表明,该方法能够满足实时性的要求.     

射线源平移扫描 CT 解析重建算法研究

微焦CT通过圆周扫描可对处于视场内的物体实现高分辨力成像。针对超出视场的大物体CT成像,一种射线源平移扫描CT(STCT)被提出。STCT采用射线源直线运动的方式采集投影数据,具有结构简单、应用灵活等特点。由于在STCT扫描过程中,每个位置的射线只能照射到部分物体,投影数据存在截断。为了提高重建效率和处理截断投影数据,提出了基于数据重组的滤波反投影重建算法(rFBP),该算法基于STCT扫描特性和X射线衰减性质,通过对置射线源采样点与探测器像元,将截断投影数据重组为全局投影数据,并在此基础上推导了rFBP算法表达式。仿真和实物实验结果验证了rFBP算法的有效性与实用性,重建时间综合缩短至SIRT算法的0.6%。rFBP算法能避免截断伪影,准确、高效地重建STCT图像。     

改善激光共焦扫描显微镜像质的方法

为解决传统光学显微镜样本上每一点的图像都受到邻近点衍射或散射光干扰的问题,研发了一套基于C#WinForm控制平台进行连续扫描方式的激光共焦扫描显微镜(LCSM)系统,并且成功地对生物细胞进行了扫描成像。针对共焦显微镜图像像质不高的问题,提出合理选取探测器针孔直径,并通过高斯低通滤波、盲解卷积的方法,确保实现高像质。实验结果表明,基于上述方法改进后的LCSM具有较高图像质量,该方法简单易行,便于实施。     

基于机器视觉的保健酒可见异物检测系统研究与开发

针对国内某企业生产的125ml玻璃瓶装药酒内可见异物的检测要求,设计了一套基于机器视觉的高速检测系统,该系统包括机械传动子系统、多类型异物成像子系统、伺服控制子系统、图像识别与处理系统等.通过利用异物在图像中运动的连续性和噪声运动无序性等特点,提出了基于序列图像差分和阀值分割的检测算法,该方法首先采用Top-hat形态学滤波抑制图像噪声,通过三帧差分提取图像中的运动目标,然后提出改进二维最大熵阀值分割算法实现了目标的快速分割,最后根据异物运动的方向性确定是否存在异物并判断出产品质量.实验结果表明,研制的系统能够有效地检测出酒液中的异物,在检测精度、误漏检率等方面满足企业要求,已成功替代了原有的人工检测.     

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.     

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.     

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700 nm）内的光谱带宽,其最小光谱带宽优于5 nm。用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor^®488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围。     

Adaptive-weighted cubic B-spline using lookup tables for fast and efficient axial resampling of 3D confocal microscopy images

Confocal laser scanning microscopy has become a most powerful tool to visualize and analyze the dynamic behavior of cellular molecules. Photobleaching of fluorochromes is a major problem with confocal image acquisition that will lead to intensity attenuation. Photobleaching effect can be reduced by optimizing the collection efficiency of the confocal image by fast z-scanning. However, such images suffer from distortions, particularly in the z dimension, which causes disparities in the x, y, and z directions of the voxels with the original image stacks. As a result, reliable segmentation and feature extraction of these images may be difficult or even impossible. Image interpolation is especially needed for the correction of undersampling artifact in the axial plane of three-dimensional images generated by a confocal microscope to obtain cubic voxels. In this work, we present an adaptive cubic B-spline-based interpolation with the aid of lookup tables by deriving adaptive weights based on local gradients for the sampling nodes in the interpolation formulae. Thus, the proposed method enhances the axial resolution of confocal images by improving the accuracy of the interpolated value simultaneously with great reduction in computational cost. Numerical experimental results confirm the effectiveness of the proposed interpolation approach and demonstrate its superiority both in terms of accuracy and speed compared to other interpolation algorithms.     

Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging

The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the ‘Scamper’. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result almost any desired pattern could be bleached (‘written’) into fluorescent samples at high definition and then imaged (‘read’) at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.     

提高激光扫描共焦显微镜反射镜扫描器定位精度的一种方法

介绍在激光扫描共焦显微镜三维扫描光路中，使用经微步细分的步进电机来完成其中的帧扫描，将MCS──51单片机引入微机控制，并采用电流补偿法来获得均匀的微细步，从而提高图象三维重建的质量。     

Enhancement of confocal microscopy images using Mueller-matrix polarimetry

A simplified procedure based on Mueller-matrix polarimetry has recently been reported as a method of retinal image improvement in a confocal ophthalmoscope [J. M. Bueno et al ., J. Opt. Soc. Am. A 24, 1337 (2007)]. Here, we have applied the technique to imaging static samples providing well-defined reflection properties. The method uses a generator of polarization states in the illumination pathway of a confocal scanning laser system. From the calculated four elements of the Mueller matrix of any sample and instrument combination, the best images defined by different metrics were constructed. For samples with specular, diffuse and mixed reflections, the best-constructed images showed an enhancement in both objective and subjective image quality compared to the original images and those obtained from frame averaging. This technique could improve microscopic imaging in many diverse fields, particularly in biomedical imaging.     

浅谈激光共聚焦显微镜使用技巧

本文简要介绍了激光扫描共聚焦（LSCM）成像原理,并以花粉为例,详细介绍了共聚焦针孔直径、光电倍增管检测器增益、激光强度、扫描速度、扫描方式、Z轴步距等重要参数设置对共聚焦成像的不同影响。探讨了正确使用LSCM的方法与技巧,如获取高质量的图像、图像保存及图像处理,以便为科技人员利用LSCM开展更多植物学与环境科学相关的研究提供参考。     

大幅宽长波红外变帧频变焦成像

为了验证变焦扫描大幅宽成像时扫描方向边缘视场图像畸变的矫正效果,并降低扫描镜控制难度。首先分析了变焦成像时扫描镜扫描速度与扫描视场角间非线性变化关系;针对扫描镜变速扫描稳定性低且控制难度大的问题,在结合探测器变帧频分析与实现的基础上,提出采用扫描镜匀角速度扫描结合探测器变帧频变焦成像方法。推导了扫描镜匀角速度扫描时,光学系统焦距、相机实时帧频与扫描成像视场角的关系式。最后基于课题组已有的可连续变焦长波红外光学系统,搭建了一套基于扫描镜扫描成像的大幅宽长波红外变焦实验系统,并利用该系统进行了成像验证。成像结果表明,采用变帧频结合变焦成像方法,可有效抑制大幅宽成像时扫描方向边缘视场图像畸变问题。变帧频成像方法降低了变焦扫描时扫描镜扫描复杂度,证明了变焦扫描方法具备图像畸变矫正能力。