微流控实时荧光聚合酶链式反应成像非均匀性的校正

综合参考标样法和定标校正法的思想,提出了一种用于校正微流控实时荧光聚合酶链式反应(PCR)系统荧光成像非均匀性的”多目标像素区域定标线性校正”算法,以提高其检测结果的准确性.以与PCR荧光标记物SYBR Green光谱特性相似的荧光素钠溶液为样本,检测了11种不同浓度的均匀荧光素钠溶液受激发射荧光信号的强度,分析了各个目标像素区域荧光强度和荧光素钠溶液浓度之间的线性响应关系,采用两点定标校正方法计算了CCD各个目标像素区域的校正系数矩阵.实验表明,3种浓度荧光素钠溶液的成像均匀度分别从校正前的71.28％、72.01％、70.73％提高到校正后的77.49％、80.07％、90.64％;微流控四腔芯片中相同浓度的DNA样品在PCR扩增阶段的Ct值相对标准偏差由校正前的4.38％、1.94％、3.31％减小到校正后的2.44％、0.79％、1.31％,显著提高了微流控实时荧光PCR检测结果的准确性.     

线扫描准共焦荧光成像

为了克服目前生物芯片荧光检测方法中诸如系统结构复杂、检测速度慢、灵敏度低、成本高等缺点,提出了一种新型生物芯片荧光检测方法——线扫描准共焦荧光成像法,并搭建了初步原理性装置。用线扫描代替共聚焦中的点扫描,将二维扫描变为一维扫描,在保持高灵敏度的同时,增加了探测速度,简化了系统,降低了成本。为了验证方法的可行性,使用搭建的原理性装置对手工点样的低密度DNA生物芯片进行了荧光成像检测。实验结果显示,系统的空间分辨率18μm,在使用像素平均法降噪后,测量浓度为0.03μmol/l的探针溶液所得信噪比为5.5×102。这项技术综合了面成像检测方法的低成本、结构简单的优势和点共焦方法具有的高分辨率的优点,适合在实验室中对生物芯片进行检测研究。     

多TDICCD拼接相机成像非均匀性实时校正的硬件实现

提出了一种基于现场可编程门阵列(FPGA)平台实现的成像非均匀性实时校正算法来解决遥感多时间延迟积分(TDI) CCD拼接相机存在的问题.首先介绍了拼接相机成像非均匀性(PRNU)的定义及其产生的原因,然后针对拼接相机的特点,提出了一种通道内用两点定标法、通道间用增益平均法、片间用自适应场景补偿法的复合非均匀性校正算法...     

多通道时间延迟积分CCD辐射标定和像元实时处理

剖析了时间延迟积分CCD(TDICCD)像元校正对信噪比和调制传递函数测试的影响,设计了基于辐射标定和地面控制的多通道TDICCD像元响应非均匀性实时处理方法以提高星上实时图像的像元响应非均匀性。该方法基于辐射标定去除图像的固定图形噪声,并校正图像奇异点;通过分析星上实时传输图像,处理奇异点像元的图像问题,实现地面控制像元响应非均匀性的实时调整。提出的系统实时处理方式可以不改变硬件结构即有效改善图像的视觉效果,对于坏像元、像元性能降低、像元污染等特定情况有很强的纠错能力。实际成像试验的对比表明,该方法对信噪比、调制传递函数有很好的修正作用,其精确调整能力分别达到0.1db及0.01;在50%饱和值下测试的图像非均匀性指标达到1.25%。该方法结构简单,在工程实践中有很好的应用前景。     

微偏振片阵列红外成像非均匀性产生机理及其校正

集成微偏振片阵列红外成像系统的偏振度图像对非均匀性高度敏感,不经非均匀校正的偏振度图像存在较大误差。为了校正微偏振片阵列红外成像的非均匀性,以入射光Stokes矢量形式,建立了光电转换基本过程的偏振像素模型,基于入射激励和辐射响应数据,分析了微偏振阵列与红外焦面联合作用下非均匀性产生机理。提出一种基于多次辐射测量的矩阵形式的非均匀性校正方法,该方法通过构造多组测量方程,求解偏振像元的增益矢量,由相邻四像元增益矢量组成超级像元的增益矩阵,结合Stokes矢量提取矩阵,逆向求解重构点的校正矩阵。实验数据表明:该方法比两点法降低非均匀性约5%~20%,有效改善红外偏振度图像质量。     

显微荧光光谱成像仪的研究与设计

将光谱分析技术快速、灵敏、准确等独特的优点,与光学成像技术的定位记录特点融合、构成光谱成像技术,可以提供分析试样中各种化学或生化成份的分布图像,因而可以获得定性、定量和定位综合、更丰富的分析信息。报导在商售落射荧光显微镜基础上,实现显微镜荧光光谱成像技术的理论基础、系统组成、设计技术关键等一系列研究工作,设计研制成了可在250~680nm波长范围内自动扫描的光纤激发显微荧光光谱成像仪样机,并获得了满意的实验结果。     

红外成像系统的非均匀性实时校正

针对红外相机系统响应非均匀性所产生的竖条纹固定噪声,提出了基于多线程优化的单帧红外图像非均匀性实时校正算法。该算法参考全变分理论的非均匀性校正算法确定噪声与图像输出之间的加性校正关系,建立了描述条纹噪声的数学模型;通过滤波算法将图像分为高频分量和低频分量,运用建立的条纹噪声模型,基于最小二乘法从高频部分中拟合出条纹噪声。最后,使用全变分理论确定的加性关系对图像进行校正。为了提高算法的计算速度,运用多线程技术对算法进行了优化。对提出的算法与MIRE(Midway Infrared Equalization)算法及传统双边滤波算法进行了图像质量评价和性能对比。结果显示:本文算法使图像非均匀性较原图降低了3%;算法效率与MIRE算法无异,系统运行时间在320×256的14位红外图像上为1.5ms/frame,达到了工程标准。     

多TDI-CCD拼接相机成像非均匀性的校正

针对多TDI-CCD拼接相机存在成像非均匀性问题,开展了对拼接相机输出图像的片内及片间综合校正算法研究.结合CCD相机特性介绍了TDI-CCD的工作原理以及拼接相机成像非均匀性的产生机理.然后,分别对拼接相机片内及片间非均匀性校正的原理进行分析,提出了片内采用两点法校正,片间采用比值平均综合法校正.最后,对片内及片间综合非均匀性校正的参数标定及校正方法进行了探讨.实验结果表明,对存在8.4%非均匀性的原拼接输出图像,采用片内与片间综合校正法校正后,图像非均匀性达到了2.7%,表明该校正方法可基本满足TDI-CCD拼接相机对成像非均性校正的要求,其算法有效实用.     

荧光共振能量转移显微术及其新进展

本文叙述荧光共振能量转移显微术及荧光寿命成像显微术的原理、方法及特点。同时介绍利用荧光共振能量转移显微术研究信号分子Rac蛋白在3T3成纤维细胞内的定位及活化过程,以及利用荧光共振能量转移—荧光寿命成像显微术研究转录因子CAATT/增强子结合蛋白α在小鼠垂体细胞内的二聚化现象。     

Flat field correction for high‐throughput imaging of fluorescent samples

Vignetting of microscopic images impacts both the visual impression of the images and any image analysis applied to it. Especially in high‐throughput screening high demands are made on an automated image analysis. In our work we focused on fluorescent samples and found that two profiles (background and foreground) for each imaging channel need to be estimated to achieve a sufficiently flat image after correction. We have developed a method which runs completely unsupervised on a wide range of assays. By adding a reliable internal quality control we mitigate the risk of introducing artefacts into sample images through correction. The method requires hundreds of images for the foreground profile, thus limiting its application to high‐throughput screening where this requirement is fulfilled in routine operation.     

Fluorescence photobleaching-based shading correction for fluorescence microscopy

A thin fluorescent test layer, which is used in a practically mono-exponential bleaching regime, is employed to determine separately the excitation intensity and the fluorescence detection efficiency distributions in the field of view of a confocal fluorescence microscope. We demonstrate that once these distributions are known, it is possible to correct an image of a specimen for intensity variations which are caused by spatial nonuniformities of the illumination and the detection efficiency of the microscope. It is indicated that, provided a photophysically well-characterized fluorescent test layer is available, the method is potentially capable of quantifying the fluorescence intensities in an image of a specimen in terms of the fluorescence quantum yield, the absorption cross-section and the concentration of the fluorophore in the specimen.     

An FFT-based method for attenuation correction in fluorescence confocal microscopy

A problem in three-dimensional imaging using a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. A new method is proposed to correct for these effects. The approach, valid for weak attenuation, consists of multiplying the measured fluorescence intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the fast Fourier transform. Analytical and numerical estimates are given for the degree of attenuation under which the method is valid, and the method is applied to various test images. A real CSLM image is restored. Finally, the method is compared with a recent iterative method with regard to numerical accuracy and computational efficiency.     

Absorption and scattering correction in fluorescence confocal microscopy

In three-dimensional (3-D) fluorescence images produced by a confocal scanning laser microscope (CSLM), the contribution of the deeper layers is attenuated due to absorption and scattering of both the excitation and the fluorescence light. Because of these effects a quantitative analysis of the images is not always possible without restoration. Both scattering and absorption are governed by an exponential decay law. Using only one (space-dependent) extinction coefficient, the total attenuation process can be described. Given the extinction coefficient we calculate within a non-uniform object the relative intensity of the excitation light at its deeper layers. We also give a method to estimate the extinction coefficients which are required to restore 3-D images. An implementation of such a restoration filter is discussed and an example of a successful restoration is given.     

Multiple frequency fluorescence lifetime imaging microscopy

The experimental configuration and the computational algorithms for performing multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) are described. The mfFLIM experimental set‐up enables the simultaneous homodyne detection of fluorescence emission modulated at a set of harmonic frequencies. This was achieved in practice by using monochromatic laser light as an excitation source modulated at a harmonic set of frequencies. A minimum of four frequencies were obtained by the use of two standing wave acousto‐optic modulators placed in series. Homodyne detection at each of these frequencies was performed simultaneously by mixing with matching harmonics present in the gain characteristics of a microchannel plate (MCP) image intensifier. These harmonics arise as a natural consequence of applying a high frequency sinusoidal voltage to the photocathode of the device, which switches the flow of photoelectrons ‘on’ and ‘off’ as the sinus voltage swings from negative to positive. By changing the bias of the sinus it was possible to control the duration of the ‘on’ state of the intensifier relative to its ‘off’ state, enabling the amplitude of the higher harmonic content in the gain to be controlled. Relative modulation depths of 400% are theoretically possible from this form of square‐pulse modulation. A phase‐dependent integrated image is formed by the sum of the mixed frequencies on the phosphor of the MCP. Sampling this signal over a full period of the fundamental harmonic enables each harmonic to be resolved, provided that the Nyquist sampling criterion is satisfied for the highest harmonic component in the signal. At each frequency both the phase and modulation parameters can be estimated from a Fourier analysis of the data. These parameters enable the fractional populations and fluorescence lifetimes of individual components of a complex fluorescence decay to be resolved on a pixel‐by‐pixel basis using a non‐linear fit to the dispersion relationships. The fitting algorithms were tested on a simulated data set and were successful in disentangling two populations having 1 ns and 4 ns fluorescence lifetimes. Spatial invariance of the lifetimes was exploited to improve the accuracy significantly. Multiple frequency fluorescence lifetime imaging microscopy was then successfully applied to resolve the fluorescence lifetimes and fluorescence intensity contributions in a rhodamine dye mixture in solution, and green fluorescent protein variants co‐expressed in live cells.     

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two‐photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy‐to‐operate platform capable to perform two‐photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

带积分作用的模糊内模时滞控制器

将Smith预估器和内模控制结构结合起来，使用模糊控制器为主控制器，并用积分环节来消除系统所存在的稳态误差，理论分析可以证明，该结构就是内模控制器，无需滤波环节就可以得到良好的鲁棒性，而模糊控制器本身的非线性和鲁棒性也可以改善系统的动态性能和鲁棒性，经仿真研究发现，只要合理调整模糊比例因子和积分因子，该方法可以在一定的模型失配情况下得到比常规IMC更好的控制品质，且无稳态误差。     

Double-pulse fluorescence lifetime imaging in confocal microscopy

A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non-steady-state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determination.