Light propagation for time-domain fluorescence diffuse optical tomography by convolution using lifetime function

Time-domain light propagation in biological tissue is studied by solving the forward problem for fluorescence diffuse optical tomography using a convolution of the zero-lifetime emission light and the exponential function for a finite lifetime. We firstly formulate the fundamental equations in a time-domain assuming that the fluorescence lifetime is equal to zero, and then the solution including the lifetime is obtained by convolving the emission light and the lifetime function. The model is a two-dimensional (2-D) 10 mm-radius circle with the optical properties simulating biological tissue for the near infrared light, and contains some inclusions with fluorophores. Temporal and spatial profiles of excitation and emission light are calculated and discussed for several models with different inclusions. The results are physically reasonable and will be used for the inverse problem of fluorescence diffuse optical tomography.     

Fluorescence lifetime imaging of intracellular calcium

Fluorescence lifetime imaging microscopy (FLIM) is a new methodology for studying the spatial and temporal dynamics of macromolecule, molecules, and ions in living cells. In FLIM image contrast is derived from the mean fluorescence lifetime at each point in a two-dimensional image. In our case the lifetime was measured by the phase-modulation method. We describe our FLIM apparatus, which consists of a fluorescence microscope, high-speed gated proximity focused MCP image intensifier, and slow-scan CCD camera. To accomplish subnanosecond time-resolved imaging, the gain of the image intensifier is modulated with a high-frequency signal, resulting in stationary phase-sensitive intensity images on the image intensifier. These images are recorded using a cooled slow-scan CCD camera and stored in an image processor. The lifetime images are created from a series of phase-sensitive images at various phase shift of the gain-modulation signal. We demonstrate calcium concentration imaging in living COS cells based on Ca²⁺-induced lifetime changes of Quin-2. The phase-angle image is mapped to the Ca²⁺ concentration image using anin vitro-determined calibration curve. The Ca²⁺ concentration was found to be uniform throughout the cell. In contrast, the intensity image shows significant spatial differences, which likely reflect variations in the thickness and distribution of probe within the cell.     

Frequency-domain lifetime imaging methods at unilever research

Fluorescence lifetime imaging methodology has been successfully implemented at Unilever Research in a frequency-domain manner. The experimental rig constructed comprises a wide-bandwidth electrooptic modulator operating on a CW argon-ion laser. The modulated excitation with a typical upper modulation frequency limit of 200 MHz falls on macroscopic samples and the resultant scattered light or fluorescence emission is then imaged onto a custom gain-modulatable image intensifier and slow-scan CCD camera combination. Phase adjustment of the image intensifier relative to the laser modulator is achieved by the RF function generator driving the intensifier. Both homodyne and heterodyne (500-Hz) strobing modes are employed to generate a double image stack (scattered light reference and fluorescence emission) comprising an image sequence as a function of instrumental phase difference. These image stacks are analyzed by Fourier least-squares methods to yield lifetime images by both phase delay and normalized demodulation. Correct operation of the apparatus is deduced from the direct imaging of a quencher-induced lifetime variation of BODIPY disulfonate over a range of concentrations. A typical industrially relevant sample, comprising an investigation of the lifetime aspects of human dental enamel autofluorescence at 50MHz modulation frequency, is given. This shows that there are real emission lifetime decreases of about 0.5 nsec in white-spot lesion areas compared to the surrounding sound enamel.     

农药荧光寿命测试系统的原理与设计

介绍荧光的产生、荧光寿命的产生机理以及荧光寿命测量的基本原理。设计了一种利用直接记录法(光子计数法)测量农药荧光寿命的测试系统。该系统针对待测样品的特性，选用了相应的脉冲光源、光学元件和半导体探测器等器件，优化了各器件的工作参数，进行了简易而又科学的模块化设计，并对西维因农药的荧光寿命在无激励光干扰情况下进行了实际测试，测得了西维因溶液在500μg／L浓度时的荧光衰减曲线和荧光寿命(0.30～0.40ns)。结果表明，该系统具有结构简易、操作方便的优点，能测量100ps级的荧光寿命，适合于对能发荧光的农药进行荧光寿命的定量测量。     

通过光致还原调制氧化石墨烯寿命并用于微纳图形制备

氧化石墨烯因其宽带可调谐的荧光发射特性已被广泛应用于荧光成像、金属离子高灵敏检测和光电器件的制备.相比于荧光强度,氧化石墨烯荧光寿命不受材料厚度和激发功率的影响,具有更为稳定和均一的特性.本文研究了在激光还原过程中氧化石墨烯荧光寿命逐渐减小的变化行为,发现了长寿命sp~3杂化结构向短寿命sp~2杂化结构的转变.通过精确控制还原时间,结合激光直写技术,在单层氧化石墨烯薄膜上实现了二维码、条形码、图形和数字等微纳图形的制备,还在多层氧化石墨烯薄膜结构上获得了多寿命多层微纳图形.这种微纳图形的制备具有灵活无掩膜、高对比和多模式的特点,可用于高密度光学存储、信息显示和光电器件制备等诸多领域.     

绿光LED激发荧光的研究

介绍了绿光LED作为荧光激发光源的可行性。使用波长为 5 30nm的超高亮度绿光LED对荧光物质四氯四溴荧光素 (C2 0 H2 Br4 Cl4 Na2 O5)的荧光发射进行了研究。设计了小巧的荧光液池 ,将四个LED分别安置于液池侧面 ,聚光后的光束交汇于液池中同一位置以提高激发光的强度 ;使用 1kHz方波调制LED ,运用分光光度计方法 ,接收到波长为 5 70nm的荧光信号     

双色荧光辐射差分超分辨显微系统研究

为了拓展荧光辐射差分(Fluorescence Emission Difference,FED)显微术的应用,使得该方法可以同时对生物样品的不同组织结构进行超分辨成像,本文对双色FED显微系统展开了研究。FED的基本原理是将实心光斑扫描得到的共焦显微图像减去空心光斑扫描得到的负共焦图像,以此获得超分辨显微图像。在对单色FED显微系统进行研究后,本文提出了一种可行的双色FED显微成像系统方案。实验结果表明,在488 nm和640 nm激发光下,该系统在荧光颗粒上分别实现了135 nm和160 nm的空间分辨率,另外也能对生物样品的不同组织进行多色同时超分辨显微成像,满足了实际应用的要求。     

基于偏振滤波图像增强和动态散斑照明的宽场荧光层析显微镜

提出了一种基于偏振滤波图像增强和动态散斑照明的新型宽场荧光层析显微镜. 该显微镜采用了一种新型的偏振滤波图像增强技术, 基于激发光与荧光偏振态的差异, 利用偏振器件滤除激发光; 并利用动态散斑照明实现宽场层析. 该荧光层析显微镜具有结构简单、低成本、响应速度快、容易操作等特点. 实验研究结果表明, 本文提出的滤波方案能够显著地提高图像质量, 利用动态散斑照明实现宽场层析具有较高的纵向分辨能力. 研究丰富了在荧光显微镜中, 从强激发光中提取弱荧光信号的技术手段, 为今后发展具有快速响应, 波长可调谐的多光谱荧光层析等高端的显微镜具有重要参考意义.     

Biochip fluorescence detection system with spatial and spectral separation

A novel optical arrangement for fluorescence detection that employs spatial separation as well as spectral filter to increase the signal to noise ratio is proposed. Using a prism and two mirrors, the elliptical laser beam of a laser diode, as an excitation light, is homogenized and the transmitted excitation light is separated from the fluorescence not to reach the collecting optics. Uncooled CCD can capture the fluorescence image of up to 40 fluorescently-labeled protein patterns without scanning or mechanical translation. This paper presents the simulation, construction and measurement results of the developed optical system. The measurements show that the combination of prism and mirrors converts the excitation light from the laser diode to uniform illumination on the specimen, and provides the separation between excitation and fluorescence light to give high signal to noise ratio. It is also possible to assay various protein concentrations ranging from 1000 to 10 ng/ml reliably. We believe that the proposed fluorescence detection system can be used to build a commercially valuable, low cost, hand-held or miniature fast detection device for point-of-care applications.     

基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

基于双音外差的电光相位调制器半波电压自校准测量方法

电光相位调制器是光纤通信系统、微波光子系统和相干光通信系统中的关键器件之一. 作为器件本征参数, 电光相位调制器的半波电压通常利用光谱方法和电谱方法进行测量. 光谱方法受到光源线宽和光谱仪分辨率限制, 测量的分辨率较低; 电谱方法则需要光电检测之前将相位调制转换成强度调制, 电谱方法的主要困难在于需要对探测器的不平坦响应进行额外校准. 提出了利用双音外差实现电光相位调制器半波电压自校准测量新方法, 该方法利用双音电光相位调制的边带与移频光载波的外差拍频, 对外差拍频信号进行频谱分析, 获得电光相位调制器的半波电压; 通过设定双音调制信号的频率关系, 克服了探测器光电转换中的不平坦频率响应, 实现了自校准测量. 该方法可扩展探测器和频谱仪的测试频率两倍以上, 节省至少一半的带宽需求. 与光谱测量方法相比, 该方法测试分辨率大幅提高且避免了光源线宽的影响; 与传统电域测量方法相比, 该方法无须额外校准, 无驱动功率和工作波长限制, 且对测试仪器带宽需求降低一半以上. 实验证实了所提方法获得的电光相位调制器半波电压的测量结果与光谱分析法获得的结果一致, 且大幅度地提高了测量范围和分辨率. 该方法提供了非常简单的电光相位调制器微波特性化分析方法, 对其他光电子器件分析也提供了参考.     

New approaches to lifetime-resolved luminescence imaging

An overview of recent developments in time-resolved imaging technology is presented. A directly modulated CCD imaging device has been developed which is suitable for frequency-domain phosphorescence measurements up to more than 400 kHz. This has been used with a recently-developed blue/UV LED source to implement a low-noise, solid-state phosphorescence/fluorescence imaging system suitable for macroscopic and microscopic imaging. For shorter lifetimes a range of new light sources has become available with higher light output and/or higher frequency modulation performance. Acoustooptic tunable filters have also been investigated as optical “light gates” both for modulation of excitation and for time-resolved imaging, and the limits of performance of these devices are discussed.     

双光子阵列点激发同时多维荧光信息的处理

五维同时荧光信息显微成像方法是一种新的荧光信息获取技术，它采用了双光子阵列点激发方式.这一方法可同时获取激发阵列点每点荧光的位置信息、荧光光谱信息和荧光寿命信息，弥补了现有荧光检测技术的不同功能信息不具有同时性的缺陷.给出了从这种技术的复合信息中提取复合光谱几何强度结构图像、不同光谱几何强度结构图像、不同光谱寿命图像的方法.提出了一种激发荧光强度修正系数矩阵方法，消除阵列点激发光强不均匀对激发荧光强弱产生的不利影响，取得明显效果.实验对实际样品做了数据采集和处理，给出图像结果，表明处理的效果良好.对存在的问题也作了讨论. 关键词： 荧光信息处理 双光子 荧光光谱 荧光寿命     

Fluorescence lifetime-enhanced spectral characterization of human serum

Frequency-domain fluorescence lifetime techniques were used for the characterization of pooled human serum, including normal serum, hyperlipid serum, and sera that had been stripped of various components. Fluorescence lifetime measurements of normal human serum revealed lifetime components primarily in the regions of 10² ps, 1–2 ns, 4–7 ns, and 9–10 ns. Phase-resolved fluorescence spectroscopy (PRFS), a frequency-domain technique that combines spectral and lifetime information, in measurements of phase-resolved fluorescence intensity (PRFI), provided the basis for comparison of the various sera. Measurements of PRFI vs excitation wavelength and emission wavelength yield a phase-resolved excitation-emission matrix (PREEM) at a given modulation frequency. Multifrequency measurements yield a three-way excitation-emission-frequency array. The multifrequency PREEMs of the various sera were compared with each other and with the corresponding two-way excitation-emission matrices (EEMs) that are obtained using conventional, steady-state fluorescence spectroscopy. Application of matrix-based analysis techniques to the steady-state and PRFS data arrays allowed direct comparison between the two approaches. Results demonstrate the enhanced discrimination among samples that is achieved through the additional dimension of fluorescence lifetime in PRFS.     

InGaP光学微盘的显微荧光图像观测及分析

利用一套ＣＣＤ显微荧光图像观测和采集分析系统,分别在室 液氮温度下对半径为５μｍ发射波长０．６５μｍ的ＩｎＧａＰ半导体光学微盘的光图像进行了观测。     

Nonlinear optical microscopy for drug delivery monitoring and cancer tissue imaging

A multimodal nonlinear optical microscope that combines coherent anti‐Stokes Raman scattering (CARS), two‐photon excitation fluorescence (TPEF), second‐harmonic generation (SHG) and sum‐frequency generation (SFG) was developed and applied to image breast cancer tissue and MCF‐7 cells as well as monitoring anticancer drug delivery in live cells. TPEF imaging showed that drugs are preferentially localized in the cytoplasm and the nuclear envelope in resistant cells. Moreover, the extracellular matrix was observed by TPEF signals arising from elastin's autofluorescence and SHG signals from collagen fibrils in breast tissue sections. Additionally, CARS signals arising from proteins and (PO₂)⁻ allowed identification of tumors. Label‐free imaging with chemical contrast of significant components of cancer cells and tissue suggests the potential of multimodal nonlinear optical microscopy for early detection and diagnosis of cancer. Copyright © 2010 John Wiley & Sons, Ltd.     

激光诱导荧光寿命及其测量

激光诱导荧光特性的研究可用于包括心血管病在内的多种疾病的诊断。荧光发射包括光谱（频域）和时间（时域）两方面的信息，后者表现为荧光寿命。在很多情况下，测量荧光寿命是比测量光谱更为有效的诊断方法。本文从理论上讨论了荧光寿命问题，并介绍两种测量方法，可用于测量人体正常组织和病变组织的激光诱导荧光寿命     

NO2分子在440～495 nm范围内的激光诱导荧光激发谱

以 NdYAG脉冲激光器泵浦的光学参量发生器/放大器作激发光源, 获得了室温、低气压条件下, NO2分子在440～495 nm波长范围内的激光诱导荧光激发谱, 将所得谱线峰归属为NO2由基电子态X2A1态向第二电子激发态B2B1态的跃迁, 利用实验所得峰值波长计算了NO2分子B2B1态的角振动频率ωe. 通过对NO2分子B2B1→X2A1跃迁的荧光时间分辨光谱进行实验研究, 得到15 Pa气压下B2B1(0, 9, 0)振动态的能级寿命τ=49 μs. 测量了荧光寿命随气压的变化关系, 利用曲线拟合得到NO2 B2B1(0, 9, 0)振动态的自发辐射寿命τ0≈55 μs和无辐射跃迁弛豫速率常数. kq=1.2×10-9 cm3 molecule-1s-1.     

Diffuse optical imaging and spectroscopy,in vivo

Based on photon migration the new goal of diffuse optical imaging is to reveal optical contrasts in the depth of biological tissues. We discuss first the origin of contrast mechanism (absorption, fluorescence and scattering) used on diffuse optical imaging and spectroscopy. Then, various experimental approaches are described based on CW, pulsed and modulated light excitation and detection. Theoretical models which provide solutions for direct and inverse problems are presented using random walk theory. Finally two studies on breast imaging and on the use of fluorescence exogeneous markers are discussed in detail.     

Simulations of measurements of fluorescence lifetimes using noise-modulated light

At the Pattern Recognition group at the Delft University of Technology, we are working on new ways to measure fluorescence lifetimes. There are two well-known ways to measure lifetimes; the phase method and the pulse method. In the phase method fluorescent material is stimulated by sinusoidally modulated light. The emitted fluorescent light will have the same modulation frequency, but there will be a phase shift between the excitation and the emission light. Measuring this phase shift will, after some simple calculation, give the lifetime of the fluorescent material. The second method is the pulse method. Short pulses of light are used to excite the material. The emitted light is detected, and from these measurements the decay curve of fluorescent light is determined. In our research we want to use a new method that may allow us to measure a mixture of lifetimes. We want to use excitation light that is modulated by a white noise signal. We are currently building an experimental setup for these measurements. We have been working on numerical and electrical simulations to investigate the properties of noise signals. Some results of these simulations are presented in this paper.