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。     

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.     

基于数字微镜器件的共焦显微镜的设计与实验

单点式共焦显微镜由于扫描速度低,光能利用率不高逐渐被多点并行的扫描方式代替.现有的并行扫描方式同样存在一些缺点,限制了测量精度的进一步提高.本文研究了基于数字微镜器件(digital micromirror device,DMD)的多路并行共焦显微镜.利用计算机程序控制DMD芯片上微镜的偏转状态,从而对光源进行调制,形成多路光束,其作用相当于可以控制的虚拟针孔阵列.设计了基于DMD的共焦检测光路,采用分光片的方法解决了由于分光棱镜内表面反射造成的重像干扰问题.对所搭建的光路进行了深度响应曲线测试实验,并且建立了基于DMD的共焦显微镜样机.样机的测量实验结果表明:基于DMD的共焦显微镜不仅具有较高的分辨率,并且具有扫描速度快、测量范围大的优点.     

单点式位移平台激光共聚焦扫描荧光显微镜

为了获得细胞图像,利用Visual Studio C#开发了移动位移平台的控制程序,使用位移平台单点扫描的方式设计激光共聚焦扫描显微镜(laser confocal scanning microscope,LCSM)。为了获得高分辨率的位移,位移由精度可以达到1nm的压电陶瓷驱动器驱动。设计了梳状和矩形两种扫描路径,通过程序设计位移补偿的方法弥补了机械运动的偏差。利用算术平均值的数字滤波方法处理数据采集卡采集的数据以减小随机噪声的影响。实验结果证明,利用C#程序控制的单点式平台扫描LCSM具有较好地测量效果。     

激光共焦扫描显微镜的光学特性研究

研究了最新发展的激光头焦扫描显微镜的下述光学特性：分辨本领及像的反差，层析分析原理及三维构像。还给出激光共焦扫描显微镜的基本光学系统及其光路按排。     

基于数字微镜的共焦显微系统的光路设计

详细叙述了共焦技术中的横向扫描技术,介绍了基于数字微镜(DMD)的共焦显微镜结构与原理,建立了基于DMD的并行检测系统,并且进行了光路的优化设计.实验结果表明,采用传统共焦显微镜光路时,光线出射分光棱镜时存在棱镜内表面反射问题,导致DMD上同一像素块在CCD上成两个像.在此分析了上述现象产生的原理,给出了解决此问题的方...     

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。     

用径向基函数网络复原超声C扫描图像

针对超声C扫描图像中存在噪声干扰和边界模糊而导致图像质量下降的问题,提出了一种基于径向基函数网络复原超声C扫描降质图像的方法.用Φ3mm平底孔的超声C扫描降质图像对网络进行训练,建立了降质图像和复原图像之间的映射关系,并用其它降质图像验证了网络.试验结果表明,该网络能有效地消除图像中的噪声和边界模糊现象,使图像中尺寸更加接近实际尺寸.同时,通过对比三种不同网络的复原效果,得到了一个最佳网络参数.     

用径向基函数网络复原超声C扫描图像

针对超声C扫描图像中存在噪声干扰和边界模糊而导致图像质量下降的问题,提出了一种基于径向基函数网络复原超声C扫描降质图像的方法.用φ3mm平底孔的超声C扫描降质图像对网络进行训练,建立了降质图像和复原图像之间的映射关系,并用其它降质图像验证了网络.试验结果表明,该网络能有效地消除图像中的噪声和边界模糊现象,使图像中尺寸更加接近实际尺寸.同时,通过对比三种不同网络的复原效果,得到了一个最佳网络参数.     

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。     

人牙釉质的磨损机制研究

以钛合金为对摩材料,考察了人牙釉质在人工唾液介质中的滑动磨损进程,结合微观分析,研究了牙釉质的磨损机制。结果表明:在摩擦过程中,随着釉质表面发生脆性剥落,磨损从两体接触磨损转变为三体磨粒磨损,磨损加剧;随着接触时间增长,硬质磨屑尺寸变小,磨损率降低,进入稳定磨损阶段。牙釉质磨损为机械磨损过程,釉质表面基本无摩擦化学作用。     

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.     

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。     

Deconvolution improves colocalization analysis of multiple fluorochromes in 3D confocal data sets more than filtering techniques

Background and noise impair image quality by affecting resolution and obscuring image detail in the low intensity range. Because background levels in unprocessed confocal images are frequently at about 30% maximum intensity, colocalization analysis, a typical segmentation process, is limited to high intensity signal and prone to noise‐induced, false‐positive events. This makes suppression or removal of background crucial for this kind of image analysis. This paper examines the effects of median filtering and deconvolution, two image‐processing techniques enhancing the signal‐to‐noise ratio (SNR), on the results of colocalization analysis in confocal data sets of biological specimens. The data show that median filtering can improve the SNR by a factor of 2. The technique eliminates noise‐induced colocalization events successfully. However, because filtering recovers voxel values from the local neighbourhood false‐negative (‘dissipation’ of signal intensity below threshold value) as well as false‐positive (‘fusion’ of noise with low intensity signal resulting in above threshold intensities), results can be generated. In addition, filtering involves the convolution of an image with a kernel, a procedure that inherently impairs resolution. Image restoration by deconvolution avoids both of these disadvantages. Such routines calculate a model of the object considering various parameters that impair image formation and are able to suppress background down to very low levels (< 10% maximum intensity, resulting in a SNR improved by a factor 3 as compared to raw images). This makes additional objects in the low intensity but high frequency range available to analysis. In addition, removal of noise and distortions induced by the optical system results in improved resolution, which is of critical importance in cases involving objects of near resolution size. The technique is, however, sensitive to overestimation of the background level. In conclusion, colocalization analysis will be improved by deconvolution more than by filtering. This applies especially to specimens characterized by small object size and/or low intensities.     

单光源双光路激光并行共焦测量系统设计

针对传统激光并行共焦测量过程中存在的泰伯效应,提出将数字微镜器件（DMD）引入激光并行共焦测量系统来正确辨识正焦面的位置。采用了DMD作为光分束器件,从理论上验证了它是一种投影式的阵列光源,对激光分束后不会在光路方向上产生泰伯像;同时,考虑DMD不能对分束后的光线产生会聚作用,并非高效的并行光源分束器件,本文将DMD与微透镜阵列(MLA)结合构建了单光源双光路并行共焦测量系统。该系统利用DMD光路探测正焦面位置,利用微透镜阵列光路进行精确的共焦测量。实验结果表明,两种光路下的正焦面位置仅相差2 μm,在一个泰伯间距范围之内,可以较好地克服泰伯效应对激光并行共焦测量的影响,进而保证较高精度的并行共焦测量。     

Investigation of micro-cracking behaviors of human femur cortical bone during radial fretting

Radial fretting tests on human femur cortical bone with a ball-on-flat configuration were carried out in vitro under different normal contact loads. The kinetics behaviors and damage characteristics of the cortical bone were analyzed using the load versus displacement (F-D) curves. The fretting scars were examined using laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM). A nano-indenter was used to characterize the mechanical property of the osteon of the cortical bone. Morphologies showed that the primary damage form was due to micro-cracking. Around the osteon, three kinds of micro-crack configurations were observed; i.e. annular cracks—located at the concentric lamellae; radial cracks—initiated from the Harversion canal; and interstitial cracks—initiated and propagated between the interstitial lamellae. Plenty of interstitial cracks occurred in the wear scar, which indicated that the interstitial tissue was the weak zone in the structure of cortical bone. In addition, the contact stresses under the radial fretting condition were calculated by the finite element method analysis (FEMs) and the behaviors of cracking were explained. The results demonstrated that the stress concentration that occurred around the Harversian canal and osteon system resulted in a better resistance than that from the interstitial tissue. The concentric lamellae presented a better radial-fretting resistance and contact fatigue resistance than that from the interstitial lamellae.     

A high stability beam-scanning confocal optical microscope for low temperature operation

We report on the design and performance of a high stability scanning confocal microscope for optical microscopy at low temperatures. By scanning the beam in a cold objective lens system, we achieve wide fields of view without compromising image quality. Photoluminescence from single nitrogen-vacancy centers in high purity diamond is used to illustrate the imaging and stability performance of the microscope.