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.     

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。     

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.     

SIL-based confocal fluorescence microscope for investigating individual nanostructures

We developed a fluorescence confocal microscope equipped with a hemispherical solid immersion lens (SIL) and apply it to study the optical properties of light-harvesting complexes. We demonstrate that the collection efficiency of the SIL-equipped microscope is significantly improved, as is the spatial resolution, which reaches 600 nm. This experimental setup is suitable for detailed studies of physical phenomena in hybrid nanostructures. In particular, we compare the results of fluorescence intensity measurements for a light-harvesting peridinin-chlorophyll-protein (PCP) complex with and without the SIL.     

Dual-axis confocal microscope for high-resolution in vivo imaging

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle theta from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 microm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo.     

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。     

Optimized pupil-plane filters for confocal microscope point-spread function engineering

We present a new method of superresolving pupil-plane filter design in confocal microscopy in which we specify the properties of the desired point-spread function and use an optimization procedure to determine a suitable pupil-plane filter. A new, flexible method of filter implementation using reconfigurable binary optical elements is described, and experimental results are presented.     

A miniature laser speckle fluorescence sectioning microscope for cell imaging

We present a miniature fluorescence sectioning microscope which uses a diode-pumped solid-state （DPSS） laser as the light source and a fast translating diffuser to produce dynamically changing speckle patterns onto the back aperture of the objective to illuminate the sample. Optical sectioning, which originates from the statistical characteristics of laser speckles, is obtained by calculating the contrast of a series of fluorescence images. High contrast fluorescence sectioning images of bovine pulmonary artery endothelial （BPAE） cells are obtained. The image quality is similar to that of the images acquired by standard laser scanning confocal microscope （LSCM）. Compared with LSCM, the laser speckle fluorescence microscope （LSFM） presented in this letter has many advantages, such as simple configurations, low cost, compact, and easy to operate, which makes it possible to have wide spread applications in biomedicine.     

Characterization of a confocal microscope for time-resolved photon counting fluorescence

A confocal microscope setup is developed for time-resolved fluorescence measurements. It is added to a traditional cuvette time-resolved setup, with a pumped Ti-Sa light source. The temporal resolution of 37 ps (FWHM) is not degraded, in comparison with the cuvette setup also described. These setups allow both decay lifetime and anisotropy relaxation time determination. Fluorescence correlation spectroscopy (FCS) is used to determine the observation point size. When associated with the calcium probe calcium green, calcium concentration in single cells can be determined in 10 ms by simultaneous acquisition of early and late fluorescence photons.     

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。     

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。     

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.     

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.     

Fiber-based chromatic confocal microscope with Gaussian fitting method

The chromatic confocal microscopy is an effective method for displacement measurement. However, with relatively low detection efficiency, chromatic confocal systems from previous studies suffer from either a limited measuring range or an unsatisfying resolution. In this paper, a novel chromatic confocal system is proposed based on optical fiber with large diameter that is specifically chosen to allow more light to be detected, thus greatly improving the detection efficiency of the system. To accurately locate the peak wavelength of the recorded spectrum, four data processing methods are proposed and compared, within which the Gaussian fitting model is considered best for the system. A series of experiments are done to verify the feasibility, resolution and stability of the system. An applicable measuring range of 600 μm is discovered with a highly linear range of 400 μm. The system has a high resolution close to 0.10 μm with satisfying stability shown by a long-term displacement standard deviation of 0.16 μm.     

Measurement of thin coatings in the confocal microscope

The use of the confocal microscope for measurement of the thickness of thin transparent coatings, such as the varnish layer on compact discs, is described. The relationship between true and apparent thickness varies in a non-linear fashion, but intensity profiles show a good correspondence with calculated profiles. This provides the basis of a nomogram for prediction of coating thickness.     

Three-dimensional surface measurement using the confocal scanning microscope

The use of the depth discriminination property of the confocal scanning microscope for surface profiling has been adapted to provide a method of high-resolution three-dimensional surface profilometry. Measurements on a semiconductor specimen demonstrate the technique; depth variations of the order of 0.1 m are clearly resolved.     

共焦扫描光学显微镜的高分辨率

讨论了共焦扫描光学显微镜的高分辨率性质,指出共焦扫描显微镜由于采用点探测器,因而视场大大减小,信噪比大大提高,同时每幅图像逐点扫描形成,在光学系统信息能力不变的前提下,系统的空间域通带宽度增加和时域通带宽度减小。因而可成高分辨率的像,特别是其独特的深度分辨率特性使得可以实现光学断层扫描成像。给出了所研制的共焦扫描荧光显微镜所获得光学断层扫描图像