Optical sectioning in reciprocal fiber-optic based chromatic confocal microscope

A theoretical analysis for studying the optical sectioning in a chromatic confocal microscope employing the same single mode fiber for illumination and detection is described. The confocality achieved by different wavelengths due to the fixed size of the fiber core is investigated using diffraction theory. The detected signal level for the different wavelengths is also calculated. The ratio of the numerical aperture of the lens and the fiber is found to play a strong role in the performance of the system.     

Fiber-coupled multiplexed confocal microscope

We describe a new parallel scanning mechanism for confocal microscopy that is inherently fiber-optic compatible and that retains the simplicity of the line scanning confocal microscope. The method works by employing an incoherent fiber-optic bundle that maps a line illumination pattern back on itself on double passing, while separating the fibers that carry photons from out-of-focus sample planes. The transformation permits efficient rejection of out-of-focus photons by a slit aperture.     

线性色散设计的光谱共焦测量技术

研制了高精度的光谱共焦位移测量系统并完成相关测试。基于色差理论和材料优化选择设计一种色差与波长成线性关系的色散物镜,有助于平衡系统在全测量范围的灵敏度。理论分析了系统参数对系统的影响规律,计算了针孔尺寸与系统的分辨率和信噪比的关系,给出了参数优化结果。利用设计的线性色散物镜和参数优化结果,构建了光谱共焦测量系统,完成了系统的校准、测试和应用研究。结果表明,系统的轴向测量范围达到1 mm,分辨力优于0.5μm,全程测量误差小于2μm,符合设计要求。     

线性色散设计的光谱共焦测量技术

研制了高精度的光谱共焦位移测量系统并完成相关测试。基于色差理论和材料优化选择设计一种色差与波长成线性关系的色散物镜,有助于平衡系统在全测量范围的灵敏度。理论分析了系统参数对系统的影响规律,计算了针孔尺寸与系统的分辨率和信噪比的关系,给出了参数优化结果。利用设计的线性色散物镜和参数优化结果,构建了光谱共焦测量系统,完成了系统的校准、测试和应用研究。结果表明,系统的轴向测量范围达到1 mm,分辨力优于0.5 m,全程测量误差小于2 m,符合设计要求。     

数字共焦显微技术

数字共焦显微技术是一种采用数学算法来实现共焦成像的技术 ,其功能与激光共焦显微技术相同。介绍了数字共焦显微技术的原理和结构组成。与激光共焦显微技术相比 ,数字共焦显微技术具有不产生光漂白、照明激发光谱宽、易于操作使用、工作方式灵活和价格便宜的特点 ,且其成像质量好于激光共焦显微技术。因此 ,数字共焦显微技术已广泛应用于许多领域且具有广阔的应用前景。     

New prospects in dermatology: Fiber-based confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) constitutes an optical, noninvasive method providing visualization of tissue architecture with resolution similar to that of light microscopy. In dermatology, confocal imaging enables in vivo measurements of surface and subsurface skin microstructures. Skin annexes, as well as cutaneous cells from different epidermal layers, can be easily distinguished; their change in morphology from skin surface to the papillary dermis can be observed. Therefore, CSLM possesses a high potential for diagnostical purposes and dermatological research. The aspect of normal skin in contrast to the pathogenic state can be exposed. In our studies, we used in vivo fluorescence CSLM for morphometric analysis of healthy human skin and for imaging a number of clinically relevant inflammatory, proliferative, and neoplastic skin disorders. We report the ability to produce high-resolution histoimages of normal and pathological epidermis using this nondestructive visualization technique. Changes in keratinocyte size, shape, and morphology, as well as changes in the distribution pattern of the fluorescent emission of the dye, can be detected. Furthermore, novel fiber optic elements support a flexible handling of the rigid microscopic gadgetry. Four clinical examples of implementation were elected and instanced for demonstration.     

拟合法测量高斯光束的束腰半径

从高斯光束的横向光强分布特性出发,建立了小孔光阑透过功率与孔径的关系式.采用2种不同拟合方法对高斯光束的束腰半径进行了测量.为减小激光器功率不稳定带来的不利影响,实验中采用双光路进行测量.结果表明,用拟合法测量高斯光束的束腰半径不仅合理,而且可行.     

Laser confocal microscope with wavelet-profiled point spread function

We report a laser-scanning confocal reflectance microscope with a wavelet-profiled point spread function (PSF) for rapid multi-resolution extraction and analysis of microscopic object features. The PSF is generated via holography by encoding a π-phase shifting disk unto a collimated laser beam via a phase-only spatial light modulator (SLM) that is positioned at the pupil plane of the focusing objective lens. Scaling of the transverse PSF distribution is achieved by selecting a suitable ratio of the π-phase shifting disk radius and the pupil aperture radius. With one and the same objective lens and one SLM to control the phase profile of the pupil function, we produce amplitude PSF distributions that are accurate scaled representations of the circularly-symmetric Mexican hat mother wavelet.     

Mechanical scanner-less multi-beam confocal microscope with wavefront modulation

Optical Review - We propose a novel full-electronically controlled laser confocal microscope in which a liquid-crystal-on-silicon spatial light modulator and a custom CMOS imaging sensor are...     

Micromachined transmissive scanning confocal microscope

We have developed a miniature scanning confocal microscope that uses electrostatically actuated microlenses for focusing and scanning. Objective lenses, scanners, a pupil, and a pinhole of the confocal microscope are microfabricated and integrated into a volume smaller than 2 mm3 by stacking these components. Objective lenses are composed of two vertically cascaded polymer microlenses integrated into micromachined comb actuators. Raster scanning is implemented by electrostatically actuating each microlens in orthogonal directions. We have demonstrated reflection confocal imaging with 3-microm transverse resolution over a 100-microm field of view and a 0.38-mm working distance at lambda = 633 nm.     

Dual-wedge scanning confocal reflectance microscope

A confocal reflectance microscope has been developed that incorporates a dual-wedge scanner to reduce the size of the device relative to current raster scanning instruments. The scanner is implemented with two prisms that are rotated about the optical axis. Spiral and rosette scans are performed by rotating the prisms in the same or opposite directions, respectively. Experimental measurements show an on-axis lateral resolution of 1.6 microm and optical sectioning of 4.7 microm, which compares with a diffraction-limited resolution of 0.8 and 1.9 microm, respectively.     

Tracking a single fluorescent molecule with a confocal microscope

We consider the problem of tracking a single fluorescent molecule in both two and three dimensions using a confocal laser scanning microscope. An estimate of the position of the molecule is generated from the measured fluorescence signal through the use of parameter estimation theory. This estimate is used in a nonlinear controller designed both to track the position of the molecule and to provide good measurements for use in the estimation algorithm. The performance of the approach is investigated through numerical simulation for molecules undergoing diffusion and directed transport and the capabilities of the controller relative to experimental limitations are discussed.     

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。     

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。     

基于彩色共焦的位移测量系统研究

为实现小型化、非接触式、快速高精度的位移测量,设计并搭建了基于光学彩色共焦原理的位移测量系统。系统中数据拟合采用的方法是,对光谱响应函数极值周围的数据进行高斯叠加拟合,以获得光谱响应曲线的峰值,从而得到峰值所对应的波长。经过实验验证,系统的测量范围为2mm,测量精度可以达到10μm,线性度为3.4%,光谱响应的半高宽(FWHM)为49nm。因此能够满足一定精度的位移测量需求。     

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.     

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     

光谱共焦测量系统中的色散透镜组设计

光谱共焦位移测量系统具有精度高、质量轻、体积小和灵敏高等优点,可以实现在各种极端情况下的微位移测量。在光谱共焦测量系统中色散透镜组的色散特性决定了整个测量系统的性能。对色散透镜组的测量需求进行了分析,提出了详细的设计指标,基于折射、衍射两种方法对透镜组进行了设计和优化,给出了设计结果。色散透镜组的色散范围为2.2mm,光谱范围为500～700nm,线性度较好。利用分辨率为0.5nm的光谱仪接收光谱信息,理论上精度优于2μm。     

差动共焦式纳米级光聚焦探测系统的研究

为解决纳米级大范围的非接触测量问题 ,提出了基于差动式共焦显微技术的光聚焦探测系统。介绍了系统的工作原理和结构装置 ,对系统中的关键技术进行了优化设计 ,通过对共焦光路的差动设计 ,可以有效地抑制光源的噪声和漂移对测量结果的影响。初步实验结果表明 ,该系统的轴向分辨率可达 2 nm