荧光波长对共焦显微镜成像特性的影响

导出了共焦显微镜中不同荧光波长情况下的荧光功率传输函数、三维脉冲响应函数（3D－PSF）和三维光学传递函数（3D－OTF）。结果表明，不同的荧光波长对共焦显微镜的空间截止频率、分辨率、光学传递函数存在明显的影响。当激发波长与荧光波长的比值下降到一定程度时，可以看到明显的失锥现象。     

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…     

Three-dimensional color photon counting microscopy using Bayesian estimation with adaptive priori information

In this Letter, we propose a novel three-dimensional(3D) color microscopy for microorganisms under photonstarved conditions using photon counting integral imaging and Bayesian estimation with adaptive priori information. In photon counting integral imaging, 3D images can be visualized using maximum likelihood estimation(MLE). However, since MLE does not consider a priori information of objects, the visual quality of 3D images may not be accurate. In addition, the only grayscale image can be reconstructed. Therefore, to enhance the visual quality of 3D images, we propose photon counting microscopy using maximum a posteriori with adaptive priori information. In addition, we consider a wavelength of each basic color channel to reconstruct 3D color images. To verify our proposed method, we carry out optical experiments.     

Theoretical investigation on Raman induced kerr effect spectroscopy in nonlinear confocal microscopy

The imaging theory of Raman induced Kerr effect spectroscopy (RIKES) in nonlinear confocal microscopy is presented in this paper. Three-dimensional point spread function (3D-PSF) of RIKES nonlinear confocal microscopy in isotropic media is derived with Fourier imaging theory and RIKES theory. The impact of nonlinear property of RIKES on the spatial resolution and imaging properties of confocal microscopy have been analyzed in detail. It is proved that RIKES nonlinear confocal microscopy can simultaneously provide more information than two-photon confocal microscopy concerning molecular vibration mode, vibration orientation and optically induced molecular reorientation, etc. It is shown that RIKES nonlinear confocal microscopy significantly enhances the spatial resolution and imaging quality of confocal microscopy and achieves much higher resolution than that of two-photon confocal microscopy.     

集成成像同名像点三维形貌获取方法

为了实现被动式三维形貌获取技术, 首先利用光线追迹方法从理论上对集成成像阵列式多角度图像获取技术进行了深入分析;对于元素图像阵列中同名像点的间距和三维物点位置之间的关联性进行了理论分析;在此基础上提出了集成成像同名像点三维形貌获取方法。实验结果显示, 本文提出的集成成像同名像点三维形貌获取技术能够获取三维物体的三维形貌和任意三维点的空间坐标。定量实验结果显示获取结果相对误差小于5%, 证实了本文提出的基于集成成像同名像点三维形貌获取技术能够实现三维信息的光学获取。     

Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes

Subtractive imaging in confocal fluorescence light microscopy is based on the subtraction of a suitably weighted widefield image from a confocal image. An approximation to a widefield image can be obtained by detection with an opened confocal pinhole. The subtraction of images enhances the resolution in-plane as well as along the optic axis. Due to the linearity of the approach, the effect of subtractive imaging in Fourier-space corresponds to a reduction of low spatial frequency contributions leading to a relative enhancement of the high frequencies. Along the direction of the optic axis this also results in an improved sectioning. Image processing can achieve a similar effect. However, a 3D volume dataset must be acquired and processed, yielding a result essentially identical to subtractive imaging but superior in signal-to-noise ratio. The latter can be increased further with the technique of weighted averaging in Fourier-space. A comparison of 2D and 3D experimental data analysed with subtractive imaging, the equivalent Fourier-space processing of the confocal data only, and Fourier-space weighted averaging is presented.     

Three-dimensional optical microscopy using axially distributed image sensing

We propose three-dimensional (3D) optical microscopy using axially distributed image sensing. In the proposed method, the micro-objects are optically magnified and their axially distributed images are recorded by moving the image sensor along a common optical axis. The 3D volumetric images are generated from the recorded axial image set using a computational reconstruction algorithm based on ray backprojection. Preliminary experimental results are presented. To the best of our knowledge, this is the first report on 3D optical microscopy using axially distributed sensing.     

High Optical Magnification Three-Dimensional Integral Imaging of Biological Micro-organism

A high optical magnification three-dimensional imaging system is proposed using an optic microscope whose ocular(eyepiece) is retained and the structure of the transmission mode is not destroyed. The elemental image array is captured through the micro lens array. Due to the front diffuse transmission element, each micro lens sees a slightly different spatial perspective of the scene, and a different independent image is formed in each micro lens channel. Each micro lens channel is imaged by a Fourier lens and captured by a CCD. The design translating the stage in x or y provides no parallax. Compared with the conventional integral imaging of micro-objects, the optical magnification of micro-objects in the proposed system can enhanced remarkably. The principle of the enhancement of the image depth is explained in detail and the experimental results are presented.     

Hyperchromats with linear dependence of longitudinal chromatic aberration on wavelength

Optical lens systems that are characterized by a chromatic aberration of a predefined form are called hyperchromats. Our work describes a theory of hyperchromats with a linear dependence of longitudinal chromatic aberration on wavelength. The equations are derived for calculation of basic design parameters of these optical systems and some examples of hyperchromatic optical systems are shown. Mentioned optical systems can be used especially in 3D imaging systems and confocal microscopy.     

Reflection confocal microscope readout system for three-dimensional photochromic optical data storage

We propose to use a reflection confocal microscope (RCM) as a readout system for digital data stored in a three-dimensional (3D) photochromic optical memory. We describe the merits and the difficulties for 3D optical memory that are associated with the use of such a RCM.It is shown by means of 3D Fourier space analysis that successful reading of 3D data can be obtained by selection of the appropriate parameter for the RCM.The system parameters include the numerical aperture of the objective lens and the wavelength of the light used for analysis (reading). Experimental results of multilayer recording and reflection confocal reading in photochromic-molecule-doped poly(methyl methacrylate) are presented. Good-contrast images are obtained.     

A novel three-dimensional digital watermarking scheme basing on integral imaging

A novel integral imaging-based three-dimensional (3D) digital watermarking scheme is presented. In the proposed method, an elemental image array (EIA) obtained by recording the rays coming from a 3D object through a pinhole array in the integral imaging system is employed as a new 3D watermark. The EIA is composed of a number of small elemental images having their own perspectives of a 3D object, and from this recorded EIA various depth-dependent 3D object images can be reconstructed by using the computational integral imaging reconstruction (CIIR) technique. This 3D property of the EIA watermark can make a robust reconstruction of the watermark image available even though there are some data losses in the embedded watermark by attacks. To show the robustness of the proposed scheme against attacks, some experiments are carried out and the results are discussed.     

Three-dimensional polarimetric integral imaging

A three-dimensional (3D) polarimetric image sensing and display technique based on integral imaging is proposed. Three-dimensional polarization distribution of reflected light from a 3D object can be measured as elemental image arrays by a rotating linear polarizer. After the measurement of the polarization of the 3D object, the 3D polarimetric object can be reconstructed optically by displaying the polarization-selected elemental images in spatial light modulators with two quarter-wave plates. Experimental demonstration of 3D polarimetric imaging of a 3D object attached to two orthogonal linear polarizers is presented. To the best of our knowledge, this is the first report on 3D polarimetric sensing imaging and 3D optical reconstruction by integral imaging.     

改善共焦系统轴向分辨率的位相型光瞳滤波器

利用约束全局优化算法———CGO算法,设计了两种用于共焦系统的三区位相型光瞳滤波器.第一种滤波器在不改变系统的横向分辨率的同时,可以大幅度地提高轴向分辨率,提高了系统的层析能力.第二种滤波器在提高系统轴向分辨率的同时,又能提高其横向分辨率,适用于系统的三维成像. 关键词： CGO算法 光瞳滤波器 超分辨     

Resolution-enhanced 3D image correlator using computationally reconstructed integral images

In this paper, a novel resolution-enhanced three-dimensional (3D) image correlator using the computationally reconstructed integral images is proposed in order to extract target object’s 3D location data in a scene. Elemental images of the reference and target objects are picked up by lenslet arrays and using these elemental images, reference and target plane images are reconstructed on the output plane by means of a modified computational integral imaging reconstruction technique. Then, through cross-correlations between the reconstructed reference and the target plane images, 3D location data of the target object can be extracted from the correlation outputs. With the purpose of showing the feasibility of the proposed method, some computational and optical experiments on the target objects in space are carried out and the results are presented.     

集成成像大纵深物体的三维形貌获取技术

为了满足现代工业所需的大纵深物体的三维形貌获取需求,解决传统结构光三维形貌获取技术纵深较小的问题, 借助集成成像这种阵列式多视点获取技术,构建了基于集成成像的大纵深物体的三维形貌获取技术。从集成成像原理出发,分析了集成成像三维物点和同名像点之间的关系,得到集成成像光学获取系统参数和三维物体纵深极限之间的关系。在此基础上,利用相机和电动平移台构建了扫描式相机集成成像三维形貌获取系统,并对纵深从600 mm到3 600 mm相对独立的2个物体构建的大纵深三维物体进行了形貌获取。光学实验结果显示,该集成成像大纵深物体三维形貌获取技术能够单次获取纵深为3 600 mm的三维物体的三维形貌,为大纵深物体的三维形貌获取提供了技术支持。     

Computational integral imaging reconstruction scheme of far 3D objects by additional use of an imaging lens

In this paper, we propose a novel performance-enhanced computational integral imaging reconstruction (CIIR) scheme by additional use of an imaging lens. In the proposed scheme, elemental images can be obtained by using a simultaneous pickup scheme of far three-dimensional (3D) objects from the lenslet array in both real and virtual image fields. And additional imaging lens produces an image shift effect of 3D objects located far away from the lenslet array and improve the visual quality of reconstructed images in CIIR by overcoming limitation of pickup range in integral imaging. To show the usefulness of the proposed system, some experiments are carried out for real 3D objects and its results are presented.     

基于人眼视觉的集成成像三维显示分辨率的比较

为了对不同集成成像系统三维显示应用中的视觉分辨效果进行表征, 提出了一种基于人眼视觉的集成成像三维显示分辨率的分析比较方法. 通过分析集成成像三维显示系统的分辨率与人眼在最佳显示观看距离下分辨本领的关系, 定义了相对分辨率参数, 分析了其与集成成像三维显示实际观看时视觉分辨效果的关系. 研究结果表明, 透镜阵列的大小对集成成像三维显示的视觉分辨效果有重要的影响. 针对两个系统实例的理论计算结果表明, 系统间的相对分辨率参数差异是传统分辨率差异的1.75倍, 实验结果与理论分析一致. 该研究方法对三维显示分辨率的评价有一定的指导意义.     

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.     

The influence of the numerical aperture of a beam probing an object on the determination of the thickness of a layered object in confocal microscopy

The influence of the numerical aperture of a light beam probing an object on the value measured with a confocal interference microscope when determining the thickness of layered object has been theoretically and experimentally investigated. The dependence of the measured value on the numerical aperture in the form of an analytical formula is obtained. It is established that the measured value is determined by the average longitudinal spatial frequency of the field probing the object. The results of experimental verification of the dependence obtained allow us to conclude that it is possible to increase the measurement accuracy of geometrical thickness and refractive index of layered micro-objects by the methods of confocal microscopy.