Image formation in structured illumination wide-field fluorescence microscopy

We present a theoretical analysis of the image formation in structured illumination wide-field fluorescence microscopy (SIWFFM). We show that the optically sectioned images obtained with this approach possess the optical sectioning strengths comparable to those obtained with the confocal microscope. We further show that the transfer function behaviour is directly comparable to that of the true confocal instrument. The theoretical considerations are compared with and confirmed by experimental results.     

探头对共聚焦内窥成像系统层析能力的影响

根据透镜成像规律和单模光纤的传输特性,获得基于单模光纤的共聚焦内窥成像系统轴向光强分布,发现系统的成像过程由于单模光纤的介入呈现非线性,照明光路系统和接收光路系统仅影响系统接收信号的总强度,系统层析能力取决于探头和单模光纤性能.通过建立的共聚焦内窥成像实验装置,测量出在三组不同物镜组合情况下系统的归一化轴向光强分布.结果显示已建立的共聚焦内窥成像系统的理论模型是正确的,扫描光点的定位精度对系统层析能力影响很大,系统设计应该综合考虑光学、机械和电子方面的性能要求.     

一种新的生物医学用快速实时低相干显微成象原理

本文讨论了一种将共焦显微术与迈克耳逊干涉术相结合,并利用宽带低相干光源相干长度短的特点而形成的一种可对高密度非透明样品进行显微成象的方法,并将这种显微成象方法与共焦显微成象方法进行了比较,最后讨论了一种快速实时成象的原理,基于这种原理设计的仪器将为生物和医学工作者提供一种新的非侵入测量和诊断手段.     

无扫描光源调制层析成象显微镜(1)-理论

分析普通显微镜的成象过程可以看到,它不能进行层析成象的原因在于并非所有的空间频率都随着离焦量的增加而衰减.通过改装普通显微镜的照明光路,采用调制光源照明物体,从而获得物体的层析象与普通显微镜成象的叠加象,该叠加象经过快速算法运算后,可实时获得类似于共焦显微镜的层析象,最终实现层析成象.本文通过对本显微镜成象光学系统物理模型的简化,解释了该系统的层析能力和解码方法.根据分析可以发现,这种照明调制显微镜实际上是一种选择性照明显微镜,即选择有限宽度的层进行照明.     

非相干光对共聚焦显微镜层析能力的影响

在共聚焦显微镜中,通过系统的归一化轴向光强半高宽与探测器有效半径和非相干光源有效半径的关系曲线,获得非相干光源照明下系统光学层析能力的变化规律.发现当探测器和光源的有效半径都小于3时,系统具有较好的光学层析能力;其他情况下,系统光学层析能力下降且会受入射光场分布的影响.     

Method of obtaining optical sectioning by using structured light in a conventional microscope

We describe a simple method of obtaining optical sectioning in a conventional wide-field microscope by projecting a single-spatial-frequency grid pattern onto the object. Images taken at three spatial positions of the grid are processed in real time to produce optically sectioned images that are substantially similar to those obtained with confocal microscopes.     

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.     

Experimental investigation of fibre-optical confocal scanning microscopy: Including a comparison with pinhole detection

Reported in this paper are the experimental results on fibre-optical confocal scanning microscopy (FOCSM). We have performed various measurements in the FOCSM involving axial responses, edge responses, signal levels and interference axial responses in order to demonstrate the differences in confocal scanning imaging between using optical fibres and using pinholes. Experimentally measured results are compared with our theoretical predictions, revealing good agreements. It is shown convincingly that the FOCSM is a purely coherent imaging system as predicted in our previous theory.     

Optical sectioning in differential interference contrast microscopy

While developing a three-dimensional (3-D) measurement technique for the retardation-modulated differential interference contrast (RM-DIC) microscope, we encountered a problem. The problem was that the measurement range was restricted in λ because it applies weak phase approximation.To overcome this drawback, we propose a 3-D reconstruction method with z-axis scanning. This technique needs high optical sectioning, like confocal microscopy.We investigated the characteristic of optical sectioning in a DIC microscope, then we confirmed experimentally that a DIC microscope has high optical sectioning. We also confirmed that a RM-DIC microscope has higher optical sectioning.By combining the optical sectioning of a RM-DIC microscope and z-scanning, we developed a new 3-D reconstruction method. This novel technique overcomes the observed problem as the measurement range is increased to micron order.     

Improvements in confocal microscopy imaging using serrated divided apertures

Confocal microscopy with serrated divided apertures is presented. Compared with using two smooth D-shaped apertures in confocal microscopy, the serrated divided apertures can suppress the out-of-focus central bright spot, thus increasing the efficiency of rejection of scattered light. Diffraction analysis shows that the serrated apertures maintain the optical sectioning strength while attenuating the background coming from far from the focal plane. In addition, the signal to background ratio (S/B) is also improved.     

Improving contrast and sectioning power in confocal imaging by third harmonic generation in SiOx nanocrystallites

We present a new optical microscope in which the light transmitted by a sample-scanned transmission confocal microscope is frequency-tripled by SiOx nanocrystallites in lieu of being transmitted by a confocal pinhole. This imaging technique offers an increased contrast and a high scattered light rejection. It is demonstrated that the contrast close to the Sparrow resolution limit is enhanced and the sectioning power are increased with respect to the linear confocal detection mode. An experimental implementation is presented and compared with the conventional linear confocal mode.     

Full-field optical sectioning and three-dimensional localization of fluorescent particles using focal plane modulation

We present a technique for imaging fluorescent particles based on the axial modulation of the objective's focal plane position. This technique provides full-field optical sectioning and can be used to localize the fluorophores in three dimensions. We describe the technique and apply it to image 200 nm diameter fluorescent beads immobilized in a gel. We show that full-field optical sectioning is obtained and that the beads are localized with a precision of 10 nm in the transverse plane and 14 nm in the axial direction.     

一种实现共焦显微镜空间微分成像的新方法

提出了一种实现共焦显微镜空间微分成像的新方法。为了获得空间微分图像,首先利用时间分辨技术结合互补调制技术,获得两束相位相反的调制光,然后利用这两束相位相反的调制光结合共焦扫描技术,实现共焦显微镜的空间微分成像。实验表明:这种空间微分技术可以准确实现共焦显微镜的空间微分成像,从而获得成像物体的边缘轮廓和实现边缘增强。     

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.     

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.     

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.     

Optical amplitude multiplexing through parametric amplification in optical fibers

We propose a new technique that uses parametric amplification in optical fibers to combine two binary signals into a single quaternary amplitude-shift keying (4-ASK) signal. We develop a theoretical model to predict the power level distribution of the 4-ASK signals as a function of the extinction ratios of the input binary signals. Computer simulation results agree with the predictions of this theoretical model within a 0.9 dB margin. We also analyze the application of this technique to the generation of quaternary-amplitude optical packets, which are attractive for allowing the label to be transmitted in the same bandwidth and simultaneously to the payload. Our results indicate that such optical packets may be propagated through lengths comparable to those involved in metropolitan area networks.     

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.     

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     

Investigation of chatter marks on ground surfaces by means of optical methods

In order to characterise chatter marks, in this work, various ground samples are investigated by means of two different optical surface analysis techniques: by using a confocal white light microscope and an angle-resolved light scattering sensor, respectively. The latter is also applied to an industrial belt grinding process in both roughness- and waviness-modes of measuring. These in-process measurements are found in good agreement with those of visual counting. Data processing in terms of the Fourier transformation it is shown to equally well accesses the wavelength of chatter marks in both roughness- and waviness-modes. Therefore it is concluded that chatter marks occurring during the industrial belt grinding can be seen as a superposition of roughness changes and waviness.