Experimental investigation of fibre-optical confocal scanning microscopy: Including a comparison with pinhole detection

Reported in this paper are the experimental results on fibre-optical confocal scanning microscopy (FOCSM). We have performed various measurements in the FOCSM involving axial responses, edge responses, signal levels and interference axial responses in order to demonstrate the differences in confocal scanning imaging between using optical fibres and using pinholes. Experimentally measured results are compared with our theoretical predictions, revealing good agreements. It is shown convincingly that the FOCSM is a purely coherent imaging system as predicted in our previous theory.     

Wavelength division multiplexed confocal microscopy using supercontinuum

We investigate both theoretically and experimentally wavelength division multiplexed confocal imaging by using white light supercontinuum. We show that with the optimized pinhole diameter an axial resolution of 0.75 μm and detection efficiency of 80% can be achieved. In addition, we applied the axial WDM confocal system to 3D surface measurement and the result agreed well with that measured by commercially available surface profilometer. The measured sensitivity of the system is 3.25 nm. Finally, we demonstrated lateral confocal imaging by using supercontinuum. An effective lateral scanning range of 130 μm was obtained.     

一种提高共聚焦显微镜信噪比算法的研究

基于共焦显微镜的成像特点,建立了Kalman滤波算法的理论模型,把Kalman滤波方法引入到系统中,提出一种基于图像像素的Kalman滤波算法,并实现了实时化的Kalman滤波器。实验结果表明:该算法能够有效地提高共焦显微镜信噪比,但是以牺牲时间为代价,提高系统分辨率的根本方法还是要着重考虑优化成像系统光路和探测电路。     

一种实现共焦显微镜空间微分成像的新方法

提出了一种实现共焦显微镜空间微分成像的新方法。为了获得空间微分图像,首先利用时间分辨技术结合互补调制技术,获得两束相位相反的调制光,然后利用这两束相位相反的调制光结合共焦扫描技术,实现共焦显微镜的空间微分成像。实验表明:这种空间微分技术可以准确实现共焦显微镜的空间微分成像,从而获得成像物体的边缘轮廓和实现边缘增强。     

激光分光瞳共焦显微镜系统研制

为了满足生物类等样品对大工作距和高分辨率共焦显微镜的需求,将分光瞳技术与激光共焦显微技术结合应用到成像系统上。阐述了激光分光瞳共焦显微成像原理,首次成功搭建了相应的显微镜成像测量系统。理论分析和实验表明:分光瞳共焦显微技术独特的非共轴结构使系统的轴向分辨力是相同数值孔径物镜单轴系统的3倍以上,对理论高度为100nm的台阶样品进行成像测试,得到的样品三维形貌,成像质量良好。     

双色荧光辐射差分超分辨显微系统研究

为了拓展荧光辐射差分(Fluorescence Emission Difference,FED)显微术的应用,使得该方法可以同时对生物样品的不同组织结构进行超分辨成像,本文对双色FED显微系统展开了研究。FED的基本原理是将实心光斑扫描得到的共焦显微图像减去空心光斑扫描得到的负共焦图像,以此获得超分辨显微图像。在对单色FED显微系统进行研究后,本文提出了一种可行的双色FED显微成像系统方案。实验结果表明,在488 nm和640 nm激发光下,该系统在荧光颗粒上分别实现了135 nm和160 nm的空间分辨率,另外也能对生物样品的不同组织进行多色同时超分辨显微成像,满足了实际应用的要求。     

Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

ItisreportedrecentlythatnonlinearopticalphenomenonofSHGandTHGhasbeenobservedinmanybiologicaltissues[16].SHGandTHGhavebeenusedtoperformthethree-dimensionalimaginginlivingtissuesandhaveattractedmuchattentionrecently.TherearemanyadvantagesofusingSHGandTHGtoperformthethree-dimensionalimaginginlivingtissues,suchasnoninvasiveandnophotobleaching,inadditiontotheimagingpropertiesofmulti-photonfluorescenceimaging[7—9].Firstly,unlikeinthesingle-andmulti-photonfluorescenceprocesses,onlyvirtualstat…     

Ultraviolet short pulses from an all-solid-state Ce:LiCAF master-oscillator power-amplifier system

We have developed an all-solid-state master-oscillator-power-amplifier system employing Ce:LiCAF, a new degradation-free tunable ultraviolet laser medium pumped by the fourth harmonic of conventional 10-ns Q -switched Nd:YAG lasers. The low- Q , short-cavity Ce:LiCAF master oscillator produced a satellite-free 1-ns pulse under appropriate pumping-fluence control. 18% energy extraction with sufficient gain was achieved in a single-stage, confocal, double-pass amplifier. As a result, 289-nm, 1-ns, 14-mJ pulses were efficiently obtained from this simple laser system.     

生物样品的散射和吸收特性对双光子激发和共焦成象深度的影响

本文研究了生物样品的散射和吸收特性对双光子激发和共焦成象深度的影响.生物组织内30μm的成象结果表明,双光子激发具有比共焦成象更深的成象深度.来自生物组织的模型实验和蒙特卡罗模拟均表明,散射和吸收的影响是导致这一特点的重要原因.     

光纤共焦扫描显微术用于光盘盘基预刻槽结构的检测

基于共焦扫描差分术,提出了检测不同表面反射率的物体表面表貌的方法,并提出了在光纤共焦扫描成像术中利用边缘判据进行边缘定位的极限尺度。上述思想用于检测光盘盘基预刻槽形结构,在建立的一套光纤共焦扫描成像装置上进行了光盘盘基预刻槽结构的检测,获得了槽宽、槽间距及槽深等实验数据,与槽标准参数相符。     

共聚焦显微镜激光高速扫描控制系统设计及实现

 在传统光学扫描方法基础上,有效地将检流式与共振式光学扫描振镜结合起来,提出一种速度可达4 M/s采样率的高速激光扫描方法,并基于单片机系统设计搭建了系统控制硬件平台,编写了上位机端和下位机端应用软件。实验结果表明:该控制系统扫描速度快,性能稳定可靠,能够应用于共聚焦显微镜,实现实时扫描成像 。     

Investigation of the reflection-type double-pass confocal microscope with a phase-conjugate mirror

New experimental results about a reflection-type double-pass confocal microscope with a phase-conjugate mirror are presented. Exploiting the change of polarization of the light, when it is reflected at the object, gives the possibility to enhance the axial resolution of the microscope compared to that of the conventional confocal microscope by a factor of 65%. Furthermore, investigations on the time behavior of the PCM are presented. Received: 11 January 1999 / Revised version: 25 January 1999 / Published online: 7 April 1999     

Reflection-mode photoacoustic microscopy using a hollow focused ultrasound transducer for in vivo imaging of blood vessels

A reflection-mode photoacoustic microscope using a hollow focused ultrasound transducer is developed for highresolution in vivo imaging.A confocal structure of the laser and the ultrasound is used to improve the system resolution.The axial and lateral resolutions of the system are measured to be ～ 32 μm and ～ 58 μm,respectively.Ex vivo and in vivo modes are tested to validate the imaging capability of the photoacoustic microscope.The adjacent vein and artery can be seen clearly from the reconstructed photoacoustic images.The results demonstrate that the reflectionmode photoacoustic microscope can be used for high-resolution imaging of micro-blood vessels,which would be of great benefit for monitoring the neovascularization in tumor angiogenesis.     

基于双螺旋点扩散函数工程的多焦点图像扫描显微

在传统共聚焦显微技术的基础上,图像扫描显微技术使用面阵探测器来代替单点探测器,结合虚拟数字针孔并利用像素重定位和解卷积图像重构算法将传统宽场显微镜的分辨率提高一倍,实现了高信噪比的超分辨共焦成像.但是,由于采用逐点扫描的方式,三维成像速度相对较慢,限制了其在活体样品成像中的应用.为了进一步提高图像扫描显微术的成像速度,本文提出了一种基于双螺旋点扩散函数工程的多焦点图像扫描显微成像方法和系统.在照明光路中,利用高速数字微镜器件产生周期分布的聚焦点阵对样品进行并行激发和快速二维扫描;在探测光路中,利用双螺旋相位片将激发点荧光信号的强度分布转换为双螺旋的形式;最终,利用后期数字重聚焦处理,从单次样品扫描数据中重构出多个样品层的超分辨宽场图像.在此基础上,利用搭建的系统分别对纤维状肌动蛋白和海拉细胞线粒体进行成像实验,证明了该方法的超分辨能力和快速三维成像能力.     

Characterization of ultrasonic imaging systems using transfer functions

The transfer functions for focussed and defocussed, coherent and confocal optical imaging systems have been applied to the equivalent ultrasonic imaging systems. The transfer functions with varying degrees of defocus were calculated to show the defocus effects for ultrasonic imaging systems. Assuming that the acoustic waves are reflected perfectly on the surface of the step edge, the theoretical line-scans for small amplitude signals across a step edge, with various degrees of defocus, were generated. The first derivative of the line-scan for a step edge is shown theoretically to yield the same impulse response as that calculated using the inverse Fourier transform applied to the original transfer function. These results show how the real and imaginary parts of the transfer functions contribute to ultrasonic image formation. A method for the experimental determination of the impulse response, and the transfer functions for the characterization of an ultrasonic imaging system, such as an acoustic microscope, is provided.     

Fluorescence lidar imaging of fungal growth on high-voltage outdoor composite insulators

Remote fluorescence imaging of fungal growth on polymeric high-voltage insulators was performed using a mobile lidar system with a laser wavelength of 355 nm. Insulator areas contaminated by fungal growth could be distinguished from clean surfaces and readily be imaged. The experiments were supported by detailed spectral studies performed in laboratory using a fibre-optic fluorosensor incorporating an optical multi-channel analyser system (OMA) and a nitrogen laser emitting radiation at 337 nm.     

不同荧光波长的双光子共焦成像分析

研究了双光子共焦显微镜中不同荧光波长对成像特性的影响,导出了不同荧光波长的三维脉冲响应函数和三维光学传递函数并进行数值计算.研究结果表明：不同荧光波长对双光子共焦显微镜的三维光学传递函数、三维脉冲响应函数和空间截止频率产生明显的影响,随着荧光波长的增大,分辨率明显下降,但不会出现单光子共焦显微镜中的失锥现象,选取适当的荧光波长进行成像,有利于进一步改善图像分辨率和成像质量.     

共焦激光扫描显微镜技术进展

共焦激光扫描显微镜已延伸到光学显微术领域，包括共焦荧光显微镜、共焦扫描光纤显微镜、彩色共焦激光显微镜、共焦光栅光学显微镜等。并很快成为半导体材料检测和生物医学诊断的重要工具。本文对国外该显微镜技术的特性和新的进展进行了综述。     

Mid-infrared generation based on a periodically poled LiNbO3 optical parametric oscillator

We report a nanosecond Nd:YVO_4-pumped optical parametric oscillator (OPO) based on periodically poled LiNbO_3 (PPLN). Tuning is achieved in this experiment by varying the temperature and period of the PPLN. The design of double-pass singly resonant oscillator (DSRO) and confocal cavity enables the OPO threshold to be lowered considerably, resulting in a simple, compact, all-solid-state configuration with the mid-infrared idler powers of up to 466mW at 3.41μm.     

A telescope illumination system for a wide-field endoscope

Endoscopic illumination systems conduct light down a built-in fibre-optic bundle to illuminate the object. Many modern endoscopes employ wide-field imaging optical systems and therefore require an illuminating beam of large divergence. This is commonly provided by placing a strong diverging lens at the far end of the fibre bundle, but it results in a poor uniformity of illumination in the object space. This paper describes the design of a telescopic illumination system to overcome this problem.