A wavefront generator for complex pupil function synthesis and point spread function engineering

We describe a simple method to produce an arbitrary complex optical field using a ferroelectric liquid crystal spatial light modulator. The system is configured so as to act as a pupil plane filter in a confocal microscope. The ability to tune the complex pupil function permits the system to be used both to modify the imaging performance by effectively engineering the point spread function as well as to remove optical aberrations present in the optical system.     

光学成像系统光学波前的高精度测试

基于扩展奈波尔-泽尼克理论,分析了不同出瞳振幅分布情况对光学系统焦面处光强分布的影响.针对光学成像系统出瞳振幅实际分布状态,提出了一种新的测试光学波前的方法,解决了相位恢复算法中出瞳振幅分布不均匀和快速傅里叶变换引入计算误差的问题.通过测评实验,对一光学系统进行了测试,获得的光学系统出瞳波前(PV)值为0.196 5λ...     

部分相干照明条件下物镜表观传递函数的测定

本文介绍了一种测量部分相干照明条件下表征物镜对基频响应的表观传递函数的方法。这种方法是将干涉仪得到的出瞳处波面的干涉图用Zernike圆多项式逼近,求得波差函数。然后根据部分相干理论计算出表观传递函数。     

Simple characterisation of a deformable mirror inside a high numerical aperture microscope using phase diversity

We present a simple and versatile scheme for characterising amplitude and phase modulation by an active element, such as a deformable mirror, in the pupil plane of a high NA microscope. By placing a mirror in the vicinity of the focal plane of the objective and recording images of the reflected focal spot on a camera, we show that reliable measurements of the influence function of the mirror actuators in the pupil plane of the objective can be obtained using an iterative electric field retrieval algorithm. Compared to direct wavefront sensors, the proposed method allows characterisation for a variety of objectives with different NA and pupil sizes without modification of the setup, requires minimal space inside the microscope, and can be used with pulsed sources such as used for multiphoton microscopy. In order to validate our method, we compare our data to the results obtained with a Shack-Hartmann wavefront sensor, and show that comparable precision is achieved.     

The theory of the direct-view confocal microscope

A theory is presented which describes imaging in both conventional and scanning microscopes. This theory embraces conventional microscopes with partially coherent source and scanning microscopes with partially coherent effective source and detector, including confocal microscopes. The theory is applicable to the direct-view confocal microscope of Petrán?, the design of which is discussed. This microscope combines the resolution and depth discrimination improvements of confocal microscopy with the ease of operation of the conventional microscope.     

数字全息干涉测量的时域和复数空间处理法

描写了一种新颖的在复数域处理数字全息干涉的方法.不同于一般的直接处理相位信息的数字相位相减法,本文提出的方法处理的是可以直接得出相位的复数信号.基于这种概念,本文提出了两种时域相位提取的算法.在这两种算法中,CCD相机感光原件的每一个像素做为一个新的独立的传感器,然后每个像素的复数信号作为时间的函数被测量和处理.这两种算法被应用于有台阶的物体的形貌检测,试验的结果证明,本文提出的算法优越于现在的数字全息通用的算法.     

用于快速形状匹配的精确型高度函数特征描述

在形状匹配过程中为了提升高度函数描述子的检索精度和对边界噪声与局部变形的鲁棒性,本文提出了一种精确型高度函数特征描述算法。首先提取目标形状外轮廓,构造轮廓采样点的精确型高度函数描述子并进行特征降维,接着利用优化后的并行动态规划进行形状匹配,最后引入形状复杂度分析提升匹配效果。基于点的几何特征显著性,提出形状精度理论,进一步分析局部形变与边缘噪声对形状特征描述的影响。在MPEG-7数据库、Swedish Leaf数据库、Tools数据库和ETH-80大型3D数据库上进行匹配实验以及在Kimia99数据库上进行抗噪实验,实验结果表明:本文提出的算法效率高,匹配时间仅为高度函数描述子的12.5%,在MPEG-7和ETH-80上的检索率最高分别为90.38%和90.07%;在Swedish Leaf和Tools上,检索精度最高分别为95.07%和94.86%,检索性能和鲁棒性均优于高度函数和其他重要算法;在添加噪声的Kimia 99上,该算法的抗噪性能优于高度函数描述子,即使在噪声水平为2.0的情况下,依旧能保持91.92%的检索率。本文提出的算法检索精度高,效率高,鲁棒性好,抗噪性强,具有较好的可扩展性,能有效地应用于形状检索领域。     

Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin

We consider various strategies for confocal imaging of human skin which seek to reduce the effects of the specimen-induced aberrations. We calculate the spherical aberration introduced by the stratified structure of skin and show how the confocal signal is affected when attempting to image at various depths within the dermis. Using simple methods it is shown how images might be improved by compensating for the induced aberration. The methods include the use of an iris to reduce the pupil area, changing the refractive index of the immersion medium and using a lens with variable coverglass correction.     

Imaging periodic surface relief structures

Because of shadowing, multiple scatter and polarization effects, the interpretation of images of gratings with fine periods, isolated deep structures, and multiple scattering volume objects is seriously complicated. In this paper a review of the methods used to model such effects is presented. Periodic surface relief gratings are of particular current importance because of the possibility of producing calibration samples using them. Several examples which illustrate electromagnetic volume effects are examined. General trends which help in validating the use of Fourier-transform-based scalar transmittance theory are then indicated. The angular spectrum approach, which can be used, together with a scatter function generated using the rigorous electromagnetic theory, to calculate coherent, partially coherent and confocal images of volume objects, is also discussed.     

Finite sized coherent and incoherent detectors in confocal microscopy

We consider the effects of finite sized coherent and incoherent detectors on the axial resolution of confocal microscopes. We adopt a high-angle vector approach which takes the polarization property of the object into account. We further consider polarization imaging and show that coherent detection has an advantage over incoherent detection in terms of the value of the extinction coefficient. The desirability of aberration correction is briefly discussed.     

计算成像技术在光学检测领域的研究进展

成像系统在利用传感器记录图像信息时,只记录了强度信息而丢失了重要的相位信息。 传统的干涉相位恢复技术由于 需要满足严格的干涉条件,在使用过程中受到一定限制。 随着计算成像技术的蓬勃发展,以强度传输方程和相干衍射成像以及 在此基础上发展起来的相干调制成像、叠层扫描相干衍射成像为代表的非干涉相位恢复技术受到广泛关注,并被运用于光学检 测领域。 这类技术无需参考光,系统结构简单,可通过衍射强度图直接获得相位信息,在检测领域有巨大的应用潜力。 基于此, 介绍了几种典型的计算成像技术的研究现状与最新进展,同时讨论分析了各类方法的主要技术特点。     

现代显微成像技术综述

概述了光学宽视场显微镜、共聚焦显微镜、超分辨率显微镜中所应用的现代显微成像技术,对各种传统和先进的显微成像原理进行了总结。光学宽视场显微镜最常用的显微技术有明场成像、暗场成像、相衬成像、偏光成像、微分干涉(DIC)成像、调制对比成像和荧光成像。相衬成像中根据不同的成像结构还有切趾相衬成像。微分干涉除了传统的偏振光照明还有圆偏振光照明(C-DIC)和专用于塑料的微分干涉(PlasDIC)。共聚焦显微镜随着计算机技术和制造技术的发展而有了巨大的发展。除了传统的共聚焦荧光显微镜以外,还有连续反斯托克斯拉曼散射(CARS)共聚焦、多光子共聚焦和白光共聚焦。超分辨率显微镜中主要介绍了受激辐射淬灭(STED)技术和紧随基态淬灭显微技术的单分子返回(GSDIM)技术。     

Image sharpness and contrast transfer in coherent confocal microscopy

Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non-confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non-confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non-confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non-confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non-confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non-confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.     

Bloch wave-based calculation of imaging properties of high-resolution scanning confocal electron microscopy

An efficient, Bloch wave-based method is presented for simulation of high-resolution scanning confocal electron microscopy (SCEM) images. The latter are predicted to have coherent nature, i.e. to exhibit atomic contrast reversals depending on the lens defocus settings and sample thickness. The optimal defocus settings are suggested and the 3D imaging capabilities of SCEM are analyzed in detail. In particular, by monitoring average image intensity as a function of the probe focus depth, it should be possible to accurately measure the depth of a heavy-atom layer embedded in a light-element matrix.     

Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy

We report on the introduction of active optical elements into confocal and multiphoton microscopes in order to reduce the sample-induced aberration. Using a flexible membrane mirror as the active element, the beam entering the rear of the microscope objective is altered to produce the smallest point spread function once it is brought to a focus inside the sample. The conventional approach to adaptive optics, commonly used in astronomy, is to utilise a wavefront sensor to determine the required mirror shape. We have developed a technique that uses optimisation algorithms to improve the returned signal without the use of a wavefront sensor. We have investigated a number of possible optimisation methods, covering hill climbing, genetic algorithms, and more random search methods. The system has demonstrated a significant enhancement in the axial resolution of a confocal microscope when imaging at depth within a sample. We discuss the trade-offs of the various approaches adopted, comparing speed with resolution enhancement.     

多维关联函数建模及其低碳实例检索应用

针对低碳设计多维度实例检索建模及其降维计算问题,提出了一种多维关联函数建模及其检索应用方法。首先,通过变斜率原理改进侧距,构建了基于改进侧距的多维侧距模型。然后,针对侧距难以准确描述设计参数与最优值之间距离的问题,建立了多维关联函数及其三层次的低碳实例检索方法。最后以螺杆空压机为例进行了验证,结果表明了改进侧距的正确性和有效性、多维关联函数降维计算的快速性与有效性,以及低碳实例检索的合理性与实例间的差异性。     

Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive‐index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high‐end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive‐index mismatch for high‐aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high‐angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point‐scanning confocal microscope and showed that a nearly index‐matched, micron‐sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.     

Single-pinhole confocal imaging of sub-resolution sparse objects using experimental point spread function and image restoration

Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Maximum likelihood image restoration algorithm has been used in conjunction with a measure of the experimental point spread function for improving the three-dimensional imaging of subresolution sparse objects. The experimental point-spread-function profiles have been improved by a factor of 1.95 in lateral direction and 3.75 in axial direction resulting in full-width half maximum (FWHM) values of 91 +/- 11 nm and 160 +/- 26 nm. This amounts to 1. 43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilising a compact confocal architecture for localising punctuate fluorescent objects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restoration approach, and, of course, in the stability of the confocal system being used.