共聚焦成像系统中探测器对分辨率的影响

基于点扩展函数,推导出共焦系统的成像公式,模拟了探测器尺寸对分辨率的影响,并提出最佳针孔半径.为验证理论分析,设计并建立了一套共焦系统,测量了不同针孔时的轴向响应,并得到了鉴别率板的共焦和离焦像.     

Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector

We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning.     

Effect of an annular pupil filter on differential confocal microscopy

Combining differential confocal microscopy and an annular pupil filter, we obtained the normalized axial intensity distribution curve of an optical system. We used the sharp slopes of the axial response curve of the optical system to measure the surface profile of a reflection grating. Experimental results prove that this method can extend the axial dynamic range and improve the transverse resolution of three-dimensional profilometry by sacrificing axial resolution.     

Improvement of confocal microscope performance by shaped annular beam and heterodyne confocal techniques

In order to further improve the performance of a confocal microscope (CM) used for measurement of surface profiles and 3D microstructures, a shaped annular beam heterodyne confocal measurement method based on annular pupil filter technique and reflection confocal microscopy, is proposed to expand the measurement range and to improve the defocused property of CM. The approach proposed uses a confocal dual-receiving light path arrangement and a heterodyne subtraction of two signals received from detectors with axial offset to enable CM to be used for bipolar absolute measurement and to improve the defocused property of CM, and it uses the annular pupil filter technique to produce a binary optical shaped annular beam, which expands the measurement range by expanding the full-width at half-maximum of intensity curve received from two detectors in a heterodyne confocal microscopy system. Theoretical analyses and experimental results indicate that a shaped annular beam heterodyne microscope has a measurement range expanded from 4 to 14 μm, achieved an axial resolution of 2 nm and improved the defocused property, when ε=0.5 and NA=0.65. It can be therefore concluded that the shaped annular beam heterodyne confocal measuring method proposed is a new approach to ultraprecision measurement of surface profiles and 3D microstructures.     

Detection of Volume Changes in Calcein-Stained Cells Using Confocal Microscopy

Calcein is an intracellular fluorescent probe that has been used as an indicator of cell volume in several previous studies. These studies have reported two different fluorescence responses depending on the optical setup used to collect the data: wide-field microscopy has resulted in a decrease in fluorescence upon cell shrinkage, whereas confocal microscopy has been shown to yield the opposite result. In this short communication, we have investigated the effect of optical setup on detection of cell volume changes in calcein-stained endothelial cells. A confocal microscope was used to collect the fluorescence data, and the pinhole diameter was varied in order to examine the effects of optical section thickness on fluorescence response. For large pinhole diameters – which correspond to relatively thick optical sections – fluorescence intensity decreased when cells were induced to shrink. In contrast, for small pinhole diameters the fluorescence intensity increased with cell shrinkage. The transition between these two types of fluorescence responses occurred when using a pinhole diameter of 285 μm, which corresponds with an optical section thickness slightly less than the height of the cells. Our results have implications for the design and interpretation of experiments involving the use of calcein as a cell volume indicator.     

Compact confocal readout system for three-dimensional memories using a laser-feedback semiconductor laser

We present a compact confocal readout system for three-dimensional optical memories that uses the thresholding property of a semiconductor laser for feedback light. The system has higher axial resolution than a conventional confocal system with a pinhole. We demonstrate readout results for data recorded in two recording layers with the developed system.     

具有高空间分辨力的整形环形光式差动共焦测量法

提出一种新的具有高空间分辨力的整形环形光式差动共焦测量方法。该方法通过整形环形光式共焦测量法和锐化爱里斑主瓣，改善系统横向分辨力；通过差动共焦测量法改善系统的轴向分辨力，最终达到提高系统空间分辨能力的目的。理论分析和实验表明：整形环形光内孔归一化半径ε越大，横向分辨力改善越明显，量程扩展范围越宽；当入射光波长λ=632.8nm，物镜数值孔径取NA=0.85，ε=0.5时，该系统的横向分辨力优于0.2μm，轴向分辨力优于2nm。该方法为光触针测量系统空间分辨力的提高提供了1种新的方法，可广泛应用于超精密三维微细结构工件的超精密测量。     

Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Raman imaging. The distribution of the UV-absorbing fluorophore Hoechst 33342 in the apoptotic HeLa cells is measured in the combined cw two-photon-excited fluorescence and Raman microscopy modes. The 647-nm line of a Kr-ion laser is used to excite both the Raman scattering and the two-photon-excited fluorescence emission. The lateral and axial resolutions in the two imaging modes are compared by use of the Gaussian beam approximation and backprojection of the focal volume through the confocal pinhole.     

激光共聚焦近红外荧光扫描系统光学设计

为了实现对近红外荧光的高分辨率扫描,设计了工作在近红外光谱区的激光共聚焦光学系统。采用结构简单的凹凸双透镜物镜实现了照明光路和发射光路的设计,并采用Zemax软件进行了光学设计和仿真。实验表明:照明光路的聚焦弥散斑小于1 m,照明针孔处的聚焦光斑小于40 m,满足照明针孔的尺寸要求;发射针孔处的聚焦光斑小于10 m,满足探测针孔尺寸要求;同时照明光路和发射光路的MTF曲线的截止频率都分别满足其衍射极限分辨率的要求,照明光路在全视场空间分辨率420 lp/mm处MTF0.08,发射光路在全视场空间频率400 lp/mm处MTF0.07。     

超分辨率活体人眼视网膜共焦扫描成像系统

活体人眼共焦扫描成像系统的分辨率受到人眼像差、数值孔径和探测针孔尺度的限制,本文设计了一套超分辨活体人眼视网膜共焦扫描系统,采用自适应光学技术探测并校正人眼像差,结合光学超分辨技术提高系统分辨率,补偿有限尺度针孔对分辨率的影响,并获得活体人眼的实时、高分辨图像. 关键词： 超分辨 共焦扫描光学显微术 眼科光学 自适应光学     

基于CCD虚拟针孔探测的双轴共焦显微技术

双轴共焦显微技术具有独特的非共轴结构,与传统单轴共焦显微技术相比可利用较低数值孔径物镜实现较高的轴向分辨力,且具有工作距离大、信噪比高等优势。对基于CCD虚拟针孔(VPH)探测的双轴共焦显微成像系统的空间分辨特性进行了理论分析,并构建了相应的实验系统,对其轴向响应进行了实验验证。实验中照明物镜NAi=0.117,采集物镜NAc=0.106,θ=45°,得出系统轴向半高宽(FWHM)为2.63μm,比同等参数(NA=0.117)下单轴共焦显微系统的轴向FWHM高出约20倍。     

A shaped annular beam tri-heterodyne confocal microscope with good anti-environmental interference capability

A shaped annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a confocal microscope. It simultaneously detects far-, on-, and near-focus signals with given phase differences by dividing the measured light path of the confocal microscope into three sub-paths (signals). Pair-wise real-time heterodyne subtraction of the three signals is used to improve the anti-environmental interference capability, axial resolution, and linearity; and a shaped annular beam super-resolution technique is used to improve lateral resolution. Theoretical analyses and preliminary experiments indicate that an axial resolution of about 1 nm can be achieved with a shaped annular beam tri-heterodyne confocal microscope and its lateral resolution can be better than 0.2 $\mu $m for $\lambda =632.8$~nm, the numerical aperture of the lens of the microscope is NA $=0.85$, and the normalized radius $\varepsilon =0.5$.     

Confocal microscopy with a refractive microlens-pinhole array

The stable setup of a confocal arrangement consisting of a combination of a refractive microlens and a pinhole array is presented. The focal plane of the microlenses lies at the rear surface of the substrate in the pinhole plane. By using a microscope objective one can image the stop array onto the object at a reduced size. Surface profiles of refractive and diffractive optical elements were measured with the help of this confocal microscope.     

Enhanced background rejection in thick tissue using focal modulation microscopy with quadrant apertures

Image formation in focal modulation microscopy using quadrant apertures (QFMM) is presented. The spatial resolution is discussed: compared with confocal microscopy, QFMM can simultaneously enhance the axial and transverse resolution. In contrast to focal modulation microscopy using D-shaped apertures (DFMM), QFMM can maintain x-y symmetry of spatial resolution, thus causing less confusion in imaging interpretation. The capability of background rejection is investigated, showing QFMM has the potential to increase imaging penetration depth. The signal level is also analyzed, indicating that the preferred detector pinhole radius is greater than for DFMM, resulting in a higher signal level.     

荧光共焦扫描系统成像特性的优化

对荧光共焦扫描系统用光强点扩散函数进行傅里变换得到系统三维传递函数的数学模型，并由此求得环形透镜和各种有限大小探测器系统的光学传递函数。用计算机模拟和光学传递九数值计算，分析了采用不同环形透镜及探测器对系统成像特性的影响     

A study of the optical transfer function for annular binary filters

The annular binary pupil filters consist of two transparent annuli obstructed by an annular mask. The optical transfer functions for them are analytically derived under paraxial approximation, and three- and two- as well as one-dimensional numerical plots are presented. It is shown that by changing the parameters of transparent regions it is possible to improve the axial resolution whilst the transverse resolution remains unchanged, compared with an annular lens. Results indicate, as is well known, that this kind of filters can be applied to three-dimensional confocal microscopy.     

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.     

3D micro-inspection goes DMD

Digital image-projection and video-projection are the classical applications of Digital Micromirror Devices (DMD) but further applications in the field of optical metrology are also possible. Operated with certain patterns, a DMD can function, for instance, as an array of pinholes that may substitute the Nipkow disk presently used for lateral scanning in confocal microscopes. The various process parameters that influence the measurement (e.g. pinhole size, pinhole shape, lateral step-size and the number of pinholes used simultaneously, etc.) can be configured easily for individual measurements by simply programming the DMD. Furthermore, price-drops for metrological precision instruments may be achieved by implementation of the mass article DMD.The paper explains the basics of DMD technology, illustrates the principle setup of a newly designed instrument, shows results achieved with it and gives examples for its application.     

等宽圆环光瞳滤波器的超分辨性能研究

设计一种等宽圆环光瞳滤波器 ,并研究了它的超分辨性能。分析了光瞳中心半径和圆环数对斯特耳比、超分辨能力和旁瓣强度的影响 ,讨论了设计等宽圆环光瞳滤波器时中心半径和圆环数的优化问题。表明圆环数为奇数和偶数时 ,光瞳具有不同的超分辨性能 ;圆环数分别取 1和 2时 ,综合考虑超分辨性能较佳     

微型差动式共焦自聚焦光聚焦探测系统

为解决微小内轮廓尺寸为代表的微小尺寸的非接触式超精密测量问题，提出了将自聚焦透镜体积小的特点与共焦显微技术的高分辨率和绝对位置跟踪特性相结合的差动式自聚焦共焦微型显微技术的光探测技术，建立了相应的传感系统，介绍了系统的工作原理和构成，自聚焦透镜测头直径为1mm，两个探测器差动设置，不但消除了光源的光强漂移和探测器的电子漂移产生的共模噪声，提高了测量信噪比，而且有效地提高了系统的轴向分辨率，初步实验表明，系统轴向分辨率在倾斜率小于20度的范围内可达5nm。