Fluorescence lifetime imaging--techniques and applications

Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time‐domain FLIM by multidimensional time‐correlated single photon counting, time‐domain FLIM by gated image intensifiers, frequency‐domain FLIM by gain‐modulated image intensifiers, and frequency‐domain FLIM by gain‐modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein‐interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity‐based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM‐FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM‐based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM‐FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady‐state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the metabolic state of the cells or the tissue. By resolving changes in the binding, conformation, and composition of biologically relevant compounds FLIM delivers information not accessible by steady‐state fluorescence techniques.     

Hardware-software System for Exploring the Possibility of Application of CCD Image Sensors as Part of Gated-Viewing Systems

This paper presents the results of the development and testing of a system for studying the operating modes of CCD sensors. The purpose of the study is to verify the performance of these sensors as part of a gated-viewing system without using an image intensifier or another external fast shutter. The system can be used to control a commercial CCD sensor in an undocumented way using signals of arbitrary shape, synchronize its operation with a laser emitter, receive and digitally process an image, and transfer it to external devices. Experiments with various CCD sensors have shown that a gated-viewing surveillance system can be developed based on an interline transfer CCD sensor without an image intensifier and can be used in applications.     

Images acquisition of a high-speed boring cutter for tool condition monitoring purposes

Tool condition monitoring is an integral part of modern CNC machine control. Cutting tools status should be periodically estimated online. For the tool condition monitoring purposes, this paper provided the image acquisition of a high-speed boring cutter based on charged coupled device (CCD) camera. The image acquisition system organization and its principle of operation were introduced in detail. The transient image of boring cutter nose was acquired at a certain rotation angle position, which can be detected by angle sensor. Mathematical derivation was deduced and analyzed in order to get clear cutter nose images, which will be influenced by the optical system, CCD camera, the exposure time, and rotation speed of the boring cutter, etc. From some experiments, it can be seen that the image acquisition method based on CCD camera is effective.     

基于无衍射光的空间入射角测量及其自动标定

为了实现对空间入射角度的精密测量,基于无衍射光束的光斑中心轨迹在自由空间的传播特性,提出了一种空间入射角精密测量系统的实现方法。针对无衍射光测空间入射角测量系统难以快速、准确地建立光束空间矢量与图像传感器像素的映射关系的问题,提出了一种基于全站仪的近距直接标定方法;搭建了用于标定的系统平台,并通过全站仪的电控马达,编写了用于标定的自动控制软件;实现了对图像传感器的自动、快速标定,极大地缩减了标定时间,消除了长时间标定所引起的系统时变误差。在工作时,通过无衍射光斑特定的定中算法,获得其在图像传感器光敏面上的位置坐标,结合光束空间矢量与角度映射矩阵,通过曲面插值即可计算得出光束的空间入射角度。实验表明针对该空间入射角测量系统的标定方法可行、自动、快速且映射精度高,可适用于类似于空间角度入射角测量系统的标定。     

High speed single charge coupled device Cranz-Schardin camera

This article describes an ultrahigh speed visualization system based on a miniaturization of the Cranz-Schardin principle. It uses a set of high power light emitting diodes (LEDs) (Golden Dragon) as the light source and a highly sensitive charge coupled device (CCD) camera for reception. Each LED is fired in sequence and images the refraction index variation between two relay lenses, on a partial region of a CCD image sensor. The originality of this system consists in achieving several images on a single CCD during a frame time. The number of images is 4. The time interval between successive firings determines the speed of the imaging system. This time lies from 100 ns to 10 micros. The light pulse duration lies from 100 ns to 10 micros. The principle and the optical and electronic parts of such a system are described. As an example, some images of acoustic waves propagating in water are presented.     

一种日夜两用光学低通滤波器的研制

利用石英晶体的双折射效应和特殊红外截止滤光片设计并实现了一种日夜两用光学低通滤波器.该滤波器置于CCD摄像机传感器前可以有效地降低或消除离散光电探测器对不同空间频率目标成像所产生的拍频效应或条纹混叠现象,并能消除红外光对彩色还原的影响,从而提高了CCD摄像机成像的视觉效果.在分析光学低通滤波器原理和红外截止滤光片作用的基础上,设计了一种特殊的红外截止膜系,可使CCD摄像机白天成彩色像,夜晚成黑白像.采用双离子束溅射技术在石英晶体薄片上淀积了该膜系,经测试,光谱特性满足设计要求,成像效果理想.     

用作图法判定航空相机TDI CCD积分方向

由于Time Delay and Integration CCD(TDI CCD)是一种类似面阵结构的线阵输出CCD,其光电荷转移具有一定的方向性,所以TDI CCD图像传感器的积分方向与CCD成像运动方向必须保持一定的关系才能成像。本文以推扫成像相机和摆扫成像相机为例,介绍了一种基于高斯成像原理的作图法,用以确定TDI CCD积分方向与传感器成像运动方向的关系,分析和论证表明,TDI CCD的积分方向必须与地物在像面上的像移方向一致。 实践证明这种方法可以清楚、简便、快速地确定TDI CCD的积分方向,从而确定TDI CCD的安装方向,适宜在实际的工程设计中采用。     

利用普通CCD实现百万帧/秒超高速成像的时序驱动技术

提出了基于普通CCD的掩模式成像技术来大幅提高CCD的图像获取速度,实现百万帧/秒的CCD时序驱动方法。介绍了加掩模后CCD的工作过程,利用掩模把普通CCD的光敏区划分为带有存储区的像素阵列,实现了普通CCD的片上存储功能。分析了利用普通CCD实现百万帧/秒超高帧频的时序驱动方法,掩模形状决定了帧速,帧数和图像的分辨率。对系统的时序结构、电荷转移方式和驱动电路进行了说明,通过特殊驱动电路和图像处理软件设计实现了百万帧/秒的超高帧频。最后,对驱动时序进行了仿真和实验验证。采用条状孔阵列掩模,基于普通CCD图像传感器进行了百万帧/秒工作速度的验证,获得了14幅79pixel×79pixel的图像,其帧频达到200×104 frame/s。文章所述技术具有一定的通用性。     

Single photon x-ray detection with a CCD image sensor

Individual x rays of 5.9 and 22.4 keV have been detected and energy analyzed in single pixels of a CCD image sensor. The results indicate the CCD operates as an array of tiny Si solid state detectors providing both high spatial resolution and x-ray energy discrimination. These devices will prove useful sensors at the focus of future x-ray telescopes.     

Single Camera 3D Digital Image Correlation Using a Polarized System

In this paper, a novel single camera three dimensional digital image correlation (3D-DIC) system, using a polarized pseudo-stereo system, is proposed. Compared to traditional 3D-DIC systems using stereovision, it has a more compact structure and better vibration resistance. Compared to the conventional single camera pseudo-stereo system that splits the CCD sensor into two halves to capture the stereo views, the proposed system achieves both views using the entire CCD chip without reduction of the spatial resolution. In addition, the center of the two views stands in the center of the CCD chip, similarly to conventional 3D-DIC systems, thus minimizing the image distortion compared to the conventional pseudo-stereo system. The two overlapped views in the CCD sensor are separated using the different polarization states, and the standard 3D-DIC algorithm can be utilized directly to perform the evaluation. The principal and experimental setup are described in detail, and multiple tests are performed to validate the system.     

Characterization of a direct detection device imaging camera for transmission electron microscopy

The complete characterization of a novel direct detection device (DDD) camera for transmission electron microscopy is reported, for the first time at primary electron energies of 120 and 200 keV. Unlike a standard charge coupled device (CCD) camera, this device does not require a scintillator. The DDD transfers signal up to 65 lines/mm providing the basis for a high-performance platform for a new generation of wide field-of-view high-resolution cameras. An image of a thin section of virus particles is presented to illustrate the substantially improved performance of this sensor over current indirectly coupled CCD cameras.     

CCD图像传感器在数码速印机的应用

CCD图像传感器是近年来发展起来的新兴固体图像传感器,具有光电转移信号存储和信号传输等功能,在现代办公设备的电子扫描读取原稿、工业自动控制中的各种检测定位,图文识别和数据读取、医疗设备等领域得到广泛的应用。分析了CCD图像传感器的光电转换原理、特点和应用领域,介绍CCD图像传感器在数码速印机的应用,并对CCD图像传感器的应用发展趋势进行了展望。     

Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources

High brightness light emitting diodes are an inexpensive and versatile light source for wide‐field frequency‐domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio‐frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal‐to‐noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices (～40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal‐to‐noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.     

CCD图像灰度与照度的转换标定方法

本文提出了一种基于CCD摄像机利用图像灰度获得实际照度的新方法,该方法适用于摄像机的任意曝光和增益参数,且考虑了摄像机安装位置和角度对测量的影响。CCD摄像机照度测量法的实际应用主要涉及两个过程:一是标定过程,即应用CCD摄像机进行照度测量前,标定CCD图像灰度与照度之间的转换参数和转换模型;二是照度测量过程,即通过CCD摄像机拍摄图像,将图像灰度代入已定的转换模型,获得测量对象的照度值。其中,灰度与照度转换模型的标定是利用CCD摄像机实现照度测量的基础和关键,文中主要讨论了该模型的推导过程以及标定方法。具体为:首先通过理论推导获得图像灰度与CCD传感器感应的相对辐照度的关系,然后借助以均匀光源搭建的实验系统标定CCD传感器相对辐照度与物面实际照度之间的关系,进而获得图像灰度与物面实际照度的转换关系。在转换过程中讨论了曝光时间、增益以及CCD相机与照度测量点之间的距离和角度对转换模型的影响,为CCD摄像机照度测量法的实际应用奠定了基础。实验结果表明本测量及标定方法适用于摄像机任意曝光和增益条件,经过本方法标定后,应用CCD摄像机进行照度测量,测量结果的相对误差在4.5%以内。     

线阵CCD驱动时序及信号采集系统的设计

文中设计介绍了一种基于FPGA和ARM的线阵CCD传感器驱动时序和信号采集的实现方法。该系统通过分析TCD1707D线阵CCD的驱动时序,采用Verilog HDL硬件描述语言设计出驱动脉冲电路。CCD正常工作后,产生的模拟信号经过预处理和高速A/D转换送入FPGA的基本宏功能模块FIFO(先进先出数据缓存器),通过异步缓存实现ARM处理器对采集信号的主控及后续应用。线阵CCD驱动时序及信号采集系统,是基于CCD传感器图像处理系统的重要组成部分,经过上位测试平台验证,能够提供准确的数字图像信号。     

气室结构触觉传感器的研究

为了解决机械手触觉传感问题,介绍了一种气室结构触觉传感器及信号处理系统,并分析了传感器的结构及敏感原理和相应的信号处理方法。传感系统采用金属弹性元件为敏感器件,传感区域充满气体,当有物体触及时,弹性片将产生变形,变形信号通过光导纤维传输至信号处理器,CCD光电转换器将光信号转换为电信号。处理系统分析判断触及物体的大小、位置。阐述了传感系统所具有的体积小、触点多、灵敏度高等优点,并提出了应用设计方案。对于机械手触觉传感领域的研究具有一定的参考价值。     

TDI CCD电荷转移对遥感相机成像质量的影响

由于多相位TDI CCD行间电荷转移引起的像移无法通过光、机、电等方法消除,本文研究了电荷转移对遥感相机成像质量的影响.首先,介绍了推扫式遥感相机的工作原理与前向像移补偿方法.接着根据TDI CCD行间电荷的转移方式,利用脉冲传递函数建立了电荷转移速度模型,并对TDI CCD电荷转移速度与景物像点移动速度进行了比较分析...     

Ultrafast photon counting applied to resonant scanning STED microscopy

To take full advantage of fast resonant scanning in super‐resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue‐to‐digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (～50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time‐gated continuous wave STED technology to the usage of resonant scanning with hardware‐based time‐gating. The assembled system provides superb signal‐to‐noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant‐scanning continuous wave STED microscopy with online time‐gated detection.     

Characterisation of a standard CCD video camera devoted to the design of an efficient particle image velocimetry instrument

A particle image velocimetry (PIV) instrument using a video frame grabbing system and two unsynchronised CCD video arrays was constructed. In the optical device, the two CCD are positioned at two perpendicular axes, with an image splitter located at their intersection and a single imaging lens in front of it. An optical characterisation of each CCD array and the imaging lens is needed to ensure that the field of view is similar for each vision system. Firstly, the experimental method of Cornu was used to deduce the basic optical parameters of the imaging lens. A simple video model was then performed to extract the unknown optical parameters of the device such as the angular field of view, the efficient dimensions of each CCD array and the transfer function from the CCD to the TV screen. An identification method based on global magnification measurements allowed the resolution of the micrometric stages needed for the spatial calibration step of the final instrument, to be deduced. Experimental tests have shown that the aberration in image formation was minimum with this configuration.     

TOF-SIMS样品光学成像系统设计

本研究为飞行时间二次离子质谱仪（TOF-SIMS）设计了一种具有高空间分辨率的样品光学成像系统。该系统由一种改进的Schwarzschild双反射系统、45°反射镜、变焦镜头及CCD图像传感器构成。采用ZEMAX软件对传统Schwarzschild模型进行计算和改进,得出系统优化参数并进行仿真验证。仿真结果表明：系统最佳的成像分辨率达1 μm,极限分辨率为0.4 μm,RMS半径小于艾里斑直径,波像差满足瑞利判据,成像质量良好。