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.     

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。     

An approach to achieve lateral superresolution for small probe confocal measurement system and its element

A shaped annular beam superresolution approach is proposed to improve a lateral resolution of a small probe laser confocal measurement system (LCMS). The approach proposed enables lateral superresolution measurement of LCMS to be achieved by using a binary optical diffractive element to shape a He–Ne Gaussian laser beam into an annular beam with an inner diameter of 0.87 mm and an outer diameter of 1.8 mm required for superresolution measurement, and shift the beam spatial frequency from low to high. And a binary optical element (BOE) with 16 phase levels is designed and fabricated to shape a Gaussian laser beam into an annular beam. Preliminary experimental results indicate that an intensity distribution of a shaped annular beam is in agreement with simulation results, the diffractive efficiency is 87.2%; LCMS lateral and axial resolutions of 0.2 μm and 3 nm are achieved, respectively, and its measurement range is expanded nearly to double, when BOE is used in LCMS and , NA=0.85.     

图像复原式整形环形光横向超分辨共焦显微测量新方法

提出一种新的图像复原式整形环形光横向超分辨共焦显微测量法. 该方法首先利用二元光学器件，将高斯照明光束整形为环形光束，用于初步改善共焦显微镜的横向分辨力，然后利用基于最大似然估计法(maximum likelihood estimate, MLE)的单幅图像超分辨复原技术，重建测量图像的高频信息，来进一步改善共焦显微镜的横向分辨力. 实验表明,当λ=632.8nm，N.A. =0.85时，该方法能使共焦显微镜获得优于0.1μm的横向分辨力. 利用该方法建立的横向超分辨共焦显微系统除了具有显著的超分辨效果外 关键词： 超分辨 超分辨复原 最大似然估计 共焦成像     

Improved axial resolution in confocal fluorescence microscopy using annular pupils

The axial image of a thick fluorescent layer is studied in a confocal microscope consisting of either circular or annular pupils. The response for circular pupils is sharper than that for annular pupils if a small pinhole detector is used. But when the size of the pinhole is larger than a certain value, the response in the latter case can become sharper than that in the former. This result implies that for a given detector of finite size, axial resolution in confocal fluorescence microscopy can be improved when an annular lens is used. Our experimental results qualitatively demonstrate the theoretical prediction. The strength of optical sectioning and the axial cross-section of the three-dimensional optical transfer function are also derived from the measured data.     

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.     

Imaging biological specimens with the confocal scanning laser microscope/macroscope

A new confocal scanning laser microscope/macroscope (cslm/M) has recently been developed. It combines in one instrument the high resolution capability of a confocal scanning beam microscope for imaging small specimens, with good resolution confocal imaging of macroscopic specimens. Some of its main features include: (a) 0.25 μm lateral resolution in the microscope mode and 5 μm lateral resolution in the macroscope mode; (b) a field of view that can vary from 25 μm × 25 μm to 75,000 μm × 75,000 μm; (c) capability for acquiring large data sets from 512 × 512 pixels to 2048 × 2048 pixels; (d) 0.5 μm depth resolution in the microscope mode and 200 μm depth resolution in the macroscope mode.

In this work the cslm/M was used to image whole biological specimens (> 5 m diameter), including insects which are ideal specimens for the macroscope. Specimens require no preparation, unlike scanning electron microscope (SEM) specimens which require a conductive coating. The specimens described in this paper are too large to be imaged in their entirety by a scanning beam laser microscope, however they can be imaged by slower scanning stage microscopes. In the macroscope mode the cslm/M was used to acquire a large number (e.g. 20–40) of confocal image slices which were then used to reconstruct a three-dimensional image of the specimen. High resolution images were collected by the cslm/M by switching to the microscope mode where high numerical aperture (NA) objectives were used to image a small area of interest. Reflected-light and fluorescence images of plant and insect specimens are presented which demonstrate the morphological details obtained in various imaging modes. A process for three-dimensional visualization of the data is described and images are shown.     

Two-photon fluorescence isotropic-single-objective microscopy

Two-photon excitation provides efficient optical sectioning in three-dimensional fluorescence microscopy, independently of a confocal detection. In two-photon laser-scanning microscopy, the image resolution is governed by the volume of the excitation light spot, which is obtained by focusing the incident laser beam through the objective lens of the microscope. The light spot being strongly elongated along the optical axis, the axial resolution is much lower than the transverse one. In this Letter we show that it is possible to strongly reduce the axial size of the excitation spot by shaping the incident beam and using a mirror in place of a standard glass slide to support the sample. Provided that the contribution of sidelobes can be removed through deconvolution procedures, this approach should allow us to achieve similar axial and lateral resolution.     

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.     

A lateral super-resolution differential confocal technology with phase-only pupil filter

In order to achieve a higher lateral resolution required for ultraprecision measurement of microstructural workpieces, phase-only pupil filtering differential confocal microscopy (PFDCM), a new approach is proposed based on the differential confocal microscopy (DCM), which uses a three-zone phase-only pupil filter with lateral super-resolution capability obtained through optimized design to change the distribution of DCM three-dimensional point spread function, so that the DCM lateral resolution is therefore significantly improved while its axial resolution is slightly improved. Preliminary experimental comparison and analyses indicate that, the lateral and axial resolutions of PFDCM are better than 0.2 μm and 2 nm, respectively, when wavelength of incidence laser beam , numerical aperture of measuring lens NA=0.85, and lateral spot size with a three-zone phase-only pupil filter G_T=0.65. It is therefore concluded that PFDCM is a new approach to further improvement of lateral resolution in laser probe measurement systems.     

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.     

微分干涉差共焦显微膜层微结构缺陷探测系统

多层膜极紫外光刻掩模"白板"缺陷是制约下一代光刻技术发展的瓶颈之一,为提高对掩模"白板"上的膜层微结构缺陷的分辨能力,提出了一种微分干涉差共焦显微探测系统方案。基于标量衍射理论,计算了系统横向和轴向分辨率。利用MATLAB建模仿真,在数值孔径为0.65、工作波长为405 nm时,分析比较了微分干涉差共焦显微系统、传统显微系统和共焦显微系统的分辨率。结果表明微分干涉差共焦显微系统具有230 nm的横向分辨率和25 nm轴向台阶高度差的分辨能力(对应划痕等缺陷形式)。此外,仿真和分析了实际应用中探测器尺寸、样品轴向偏移等的影响,模拟分析了膜层微结构缺陷的探测,结果表明本系统可以探测200 nm宽、10 nm高的微结构缺陷,较另外两种系统有更好的探测能力。     

Scanning two photon fluorescence microscopy with extended depth of field

We describe the design and implementation of a light efficient binary phase-only diffraction optical element which converts a laser beam into an annular ring with the loss of less than 25% of the overall power. The use of annular illumination permits us to obtain an increase in the axial extent of the point spread function of a microscope objective without compromising the lateral resolution. The light efficiency of the system permitted two photon fluorescence images with extended depth of field to be obtained. The combination of two such extended depth of field images obtained at different parallaxes allowed the generation of high resolution two photon stereo pairs.     

超分辨在共焦三维形貌检测术中的应用

通过对共焦三维形貌检测术的分析,根据超分辨理论,在共焦系统中加入环形光瞳滤波器,以期延长系统的动态检测范围,并提高系统的横向分辨率。理论和实验结果表明该方法在适当牺牲轴向分辨率的条件下,能有效地延长系统的动态检测范围,且提高其横向分辨率。     

用于高数值孔径物镜的可调光瞳滤波器

根据矢量衍射理论，提出了一种用于提高高数值孔径显微物镜纵向分辨率的可调二元环形光瞳滤波器，通过改变滤波器的环半径获得不同的纵向分辨率，并利用共焦显微术点扩散函数的乘积性大大地降低了旁瓣相对主瓣的比值。模拟结果表明，这种新型滤波器不仅可以提高高数值孔径物镜的纵向分辨率，同时基本上不影响横向分辨率。     

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。     

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.     

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.     

Optical measurements with phase-conjugate mirrors

Phase-conjugate mirrors on the basis of photorefractive crystals offer interesting possibilities of application for optical measurement systems. In this paper we report on the application of phase-conjugate mirrors (PCMs) for optical measurement techniques. We show different schemes for realizing PCMs and discuss characteristics such as reflectivity, fidelity, and time behavior. We demonstrate that two-beam interferometers with PCMs and with a suitable phase-shifting unit in the reference arm have general advantages compared with conventional systems. It is shown that self-pumped PCMs (SPPCMs) could also be successfully used for novelty filtering systems comparing a present state of a signal phase with a reference phase. It is found that SPPCMs can improve both the axial and the lateral resolution of a confocal microscope. A 30% better resolution of the PCM system is established. Received: 14 June 1999 / Revised version: 4 October 1999 / Published online: 3 November 1999     

In vivo dark-field reflection-mode photoacoustic microscopy

Reflection-mode photoacoustic microscopy with dark-field laser pulse illumination and high-numerical-aperture ultrasonic detection is designed and implemented in noninvasively imaged blood vessels in the skin in vivo. Dark-field optical illumination minimizes the interference caused by strong photoacoustic signals from superficial structures. A high-numerical-aperture acoustic lens provides high lateral resolution, 45-120 microm in this system. A broadband ultrasonic detection system provides high axial resolution, estimated to be approximately 15 microm. The optical illumination and ultrasonic detection are in a coaxial confocal configuration for optimal image quality. The system is capable of imaging optical-absorption contrast as deep as 3 mm in biological tissue.