反射式光纤共焦扫描成像的研究

建立了反射式光纤共焦扫描成像系统,分析了光纤－集光透镜参数Ａ及物透镜有效数值孔径等对系统成像分辨率的影响。并在此基础上选择了合适参数的透镜,获得了优化的反射式光纤共焦扫描成像系统,测试结果表明,该系统具有亚微米级横向成像能力,微米级向层析能力,成像稳定性那,它将应用于材料及生物组织三维成像检测中。     

差动共焦显微成像高稳定三维扫描技术与系统

激光差动共焦显微镜具备高空间分辨率特点,但因其逐点扫描成像方式,扫描时间长,易受三维扫描系统不稳定和环境干扰等影响,产生系统漂移,影响仪器的空间分辨率。利用楔块机构高稳定特点,结合刹车机构的自由抱闸特性,设计了一种新型的轴向升降机构,由此构建了结构更具稳定特性的电动三维扫描系统。稳定性实验验证在搭建的激光差动共焦显微镜上进行,经过监测系统在90min内的轴向位置,轴向漂移小于50nm,与原三维扫描系统漂移140nm对比,漂移速度明显减慢,稳定性有显著提升,进而明显改善了差动共焦显微成像效果。     

Computation of the lateral and axial point spread functions in confocal imaging systems using binary amplitude mask

In this paper, a novel aperture based on Tolardo concept composed of a central clear disc surrounded by a series of black and white (B/W) concentric annuli of equal transmittance is presented. Different apodized apertures of different number of B/W annuli are suggested in order to improve further the three-dimensional resolving power of confocal imaging systems. Both the axial and lateral point spread functions (PSF) and the corresponding irradiances are computed in both cases of conventional and confocal scanning microscopes for the above-mentioned amplitude filters. These results of axial and lateral irradiances are graphically represented by constructing a computer program using MATLAB. The obtained results are compared with that obtained in case of circular, annular, and Martinez-Corral apodized aperture     

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.     

探头对共聚焦内窥成像系统层析能力的影响

根据透镜成像规律和单模光纤的传输特性,获得基于单模光纤的共聚焦内窥成像系统轴向光强分布,发现系统的成像过程由于单模光纤的介入呈现非线性,照明光路系统和接收光路系统仅影响系统接收信号的总强度,系统层析能力取决于探头和单模光纤性能.通过建立的共聚焦内窥成像实验装置,测量出在三组不同物镜组合情况下系统的归一化轴向光强分布.结果显示已建立的共聚焦内窥成像系统的理论模型是正确的,扫描光点的定位精度对系统层析能力影响很大,系统设计应该综合考虑光学、机械和电子方面的性能要求.     

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.     

Confocal fluorescence spectroscopy and anisotropy imaging system

We report the design and implementation of a laser scanning confocal fluorescence system with spectroscopy and anisotropy imaging capabilities. Confocal spectroscopy is achieved with a fiber pinhole that is inserted into and removed from the detection path as needed. Fluorescence anisotropy imaging is accomplished with a polarizing beam splitter placed after the conventional pinhole. Two orthogonal polarizations are detected simultaneously with balanced photomultiplier tubes. The quality of the axial sectioning that is achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and examples of polarization-sensitive fluorescence imaging are demonstrated in tumor cell monolayers.     

High-speed 3D imaging based on structured illumination and electrically tunable lens

In this Letter, we present a high-speed volumetric imaging system based on structured illumination and an electrically tunable lens(ETL), where the ETL performs fast axial scanning at hundreds of Hz. In the system,a digital micro-mirror device(DMD) is utilized to rapidly generate structured images at the focal plane in synchronization with the axial scanning unit. The scanning characteristics of the ETL are investigated theoretically and experimentally. Imaging experiments on pollen samples are performed to verify the optical cross-sectioning and fast axial scanning capabilities. The results show that our system can perform fast axial scanning and threedimensional(3D) imaging when paired with a high-speed camera, presenting an economic solution for advanced biological imaging applications.     

共焦扫描光声层析成像技术的研究

报道了一种新的光声层析成像技术－光声傅里叶变换共焦扫描成像技术，并用该技术获得了强散射物质（生物组织）的层析图像，纵向分辨能力小于0．1mm，横向分辨率小于4mm。     

采用变焦液体透镜的共焦检测系统的设计与仿真

共焦显微技术越来越广泛地应用于微机电系统和半导体器件的三维轮廓测量。设计一种新型采用变焦透镜作为轴向扫描方式的共焦显微系统,利用变焦透镜的焦距变化来代替传统的轴向位移扫描。系统实现了无机械运动的轴向扫描,消除了传统共焦系统中由位移台移动带来的振动,不仅减小了系统的复杂度,而且降低了成本。进一步设计了基于位移台和变焦透镜的两种共焦检测系统,并且对其进行了仿真实验。实验结果表明:基于位移台和基于变焦透镜的共焦系统的聚焦成像和离焦成像结果类似,并且光强的轴向分布曲线也基本相同,表明此方法可行。     

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。     

Three-dimensional integral imaging of micro-objects

We propose a method for displaying micro-objects in space that is based on three-dimensional (3D) integral imaging, in which elemental images are calculated from a two-dimensional sampling of the optical field along different depths by use of confocal scanning microscopy. Experimental results are presented to demonstrate that a uniformly magnified 3D biological specimen can be displayed in space, and thus integral imaging can be used for 3D display of confocal microscopy. To the best of our knowledge, this is the first report of 3D integral imaging of (semitransparent) micro-objects.     

Spectrally encoded slit confocal microscopy

A simple and cost-effective method for real-time imaging in confocal microscopy is proposed. Spectrally encoded slit confocal microscopy (SESCoM) uses a spectral encoding technique together with a confocal slit aperture to achieve two-dimensional images. Simulation and experimental results of the SESCoM's axial and lateral performances are presented. The measured FWHM of the axial response is 1.15 mum when an objective with a NA of 0.95 is used. FWHMs of the lateral line spread functions are measured to be 236 and 244 nm along the x and y directions, respectively. Both the axial and the lateral experimental results agree well with the simulation results.     

线结构光共焦显微成像技术的实验研究

激光共焦扫描显微镜大多采用点共焦扫描成像形式,扫描机构复杂,成本高。新型的线结构光共焦显微成像技术是对样品进行线共焦成像,只需对样品进行一维扫描就可以得到整个平面的像。设计了线结构光共焦成像实验装置,进行了线结构光共焦显微成像实验。实验结果证明了线结构光共焦成像的可行性,杂散光明显地减少,提高了成像清晰度,并且有光学层析能力。采用低照度高分辨率CCD代替价格昂贵的像增强器大大降低了系统成本。     

Talbot holographic illumination nonscanning (THIN) fluorescence microscopy

Optical sectioning techniques offer the ability to acquire three‐dimensional information from various organ tissues by discriminating between the desired in‐focus and out‐of‐focus (background) signals. Alternative techniques to confocal, such as active structured illumination, exist for fast optically sectioned images, but they require individual axial planes to be imaged consecutively. In this article, an imaging technique (THIN), by utilizing active Talbot illumination in 3D and multiplexed holographic Bragg filters for depth discrimination, is demonstrated for imaging in vivo 3D biopsy without mechanical or optical axial scanning.     

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。     

二次谐波共焦成像的分辨率

通过与双光子荧光共焦成像过程比较,研究了非线性二次谐波的强度脉冲响应函数并对它进行频域分析,获得了横向、纵向截止频率和通频带.研究结果表明：二次谐波共焦成像具有更高的分辨率.     

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。     

Improved sectioning in a slit scanning confocal microscope

We describe a simple implementation of a slit scanning confocal microscope to obtain an axial resolution better than that of a point-scanning confocal microscope. Under slit illumination, images of a fluorescent object are captured using an array detector instead of a line detector so that out-of-focus light is recorded and then subtracted from the adjacent images. Axial resolution after background subtraction is 2.2 times better than the slit confocal resolution, and out-of-focus image suppression is calculated to attenuate with defocus faster by 1 order of magnitude than in the point confocal case.     

复振幅光瞳滤波器的三维超分辨性能研究

超分辨技术中现有的纯振幅型或纯相位型光瞳滤波器,大部分只能实现轴向或横向超分辨而不能实现三维超分辨,三维超分辨在三维成像系统中有着重要的作用。因此为提高成像系统中的三维分辨能力,设计了一种复振幅光瞳滤波器,并对其三维超分辨性能进行了研究。详细分析了该光瞳滤波器的第一区半径和透射率对施特雷尔比、轴向和横向超分辨因子以及旁瓣能量的影响。通过一系列的模拟证明,借助于复振幅光瞳滤波器可以实现三维超分辨。该滤波器的优点是容易实现三维超分辨,且有比较高的施特雷尔比,缺点是三维超分辨的实现总是伴随着旁瓣能量的增加,但可以采用共焦扫描成像系统来加以抑制。