Calcium imaging using fluorescence lifetimes and long-wavelength probes

We describe imaging of calcium concentrations using the long-wavelength Ca²⁺ indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry.     

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.     

Construction and characterization of a frequency-domain fluorescence lifetime imaging microscopy system

The construction of a homodyne frequency domain fluorescence lifetime imaging microscope is described. The system consists of (i) an intensity-modulated laser excitation source, (ii) an epifluorescence microscope, (iii) a gain-modulated microchannel plate (MCP) image intensifier, and (iv) a slow-scan CCD camera. The phase and modulation homogeneity of the MCP image intensifier were determined at frequencies of 40, 100, 160, and 240 MHz. The detected modulation depths were 65, 52, 32, and 23%, respectively, and were highly homogeneously distributed. The phasedistribution image revealed iris effects at frequencies of 160 and 240 MHz but was homogeneous at lower frequencies. Lifetime imaging of a solution of the fluorescent flavoprotein lipoamide dehydrogenase demonstrated (i) the accuracy of the determined lifetimes (< 60 ps), (ii) the time resolution of the instrument (< 50 ps), and (iii) the average precision for single pixel fluorescence lifetimes (50 ps is feasible). The imaging of tiny fluorescent microspheres revealed that even in a volume of 0.3 x 10^-15 L, the standard error in the lifetimes can be as low as 79 ps. The spatial resolution of the instrument is estimated to be < 400 nm in the object plane at a 100 x magnification.     

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10^-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns). Received: 23 February 2003 / Published online: 22 May 2003 RID="*" ID="*"Corresponding author. Fax: +1-734/9361-905, E-mail: mycek@umich.edu     

Double-pass imaging polarimetry in the human eye

A Mueller-matrix imaging polarimeter was developed to measure spatially resolved polarization properties in the living human eye. The apparatus is a double-pass setup that incorporates two liquid-crystal variable retarders and a slow-scan CCD camera in the recording stage. Series of 16 images for the combinations of independent polarization states in the first and second passages were recorded for two experimental conditions: with the camera conjugated either with the retina or with the eye's pupil plane. Spatially resolved collections of Mueller matrices and the degree of polarization were calculated from those images for both retinal and pupil planes.     

Remote Estimation of the Chlorophyll Concentration of Living Trees Using Laser-induced Fluorescence Imaging Lidar

Monthly variation in chlorophyll concentration of living ginko tree leaves 65 m away from a system was remotely estimated by a laser-induced fluorescence imaging lidar. The combination of a pulsed laser and a short-time gated CCD using an image intensifier made it possible to monitor the weak fluorescence signal from the ginko tree leaves as an image. By applying the experimental idea that a ratio of intensity of the chlorophyll fluorescence at 740 nm to that at 685 nm showed a linear correlation to the chlorophyll concentration, the fluorescence image of the ginko tree obtained by the lidar was converted to the chlorophyll concentration distribution image.     

基于一代像增强器的高分辨率X射线相机

为了实现在较低剂量下获得较高的分辨率,研制了一款价格低廉的X射线CCD相机。该相机使用硫氧化钆作为转换屏、经一代像增强器增强后的图像通过光锥耦合到CCD相机上;为了提高系统的光传输效率,提出了将像增强器输出直面板或倒像器直接更换成光锥,其小端直接同CCD相机耦合的方案;通过灰度分析计算对比度曲线,拟合得出系统的本征空间分辨率为13 Lp/mm;通过拍摄实际景物,可以清晰地看到其内部的细节,显示出比较好的图像质量。     

Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca²⁺; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca²⁺ in biological samples, we demonstrate thatintensity-based measurements of Ca²⁺ with single-wavelength Ca²⁺ probes such as Calcium Crimson may falsely report the actual Ca²⁺ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.     

Fluorescence lifetime imaging with picosecond resolution for biomedical applications

We describe a novel whole-field fluorescence lifetime imaging system, based on a time-gated image intensifier and a solid-state laser oscillator-amplifier, that images lifetime differences of less than 10 ps. This system was successfully applied to discrimination between biological tissue constituents.     

低照度条件下三基色获取及真彩色融合方法研究

鉴于低照度条件下彩色成像都是采用微光、红外、紫外等波段图像融合成假彩色,提出一种利用滤光片过滤和像增强器增强的三基色获取及真彩色融合方法。采用F P滤光片设计出了透射中心在三基色光中心波长的三基色滤光片,对其光谱透过性进行分析;通过旋转三基色滤色轮将目标反射光过滤,使用像增强器对过滤后反射光增强,借助FPGA编写的控制程序实现滤色轮转速和CCD摄像机成像时间的精确同步,利用CCD摄像机获取经增强后的三基色图像,实现目标三基色图像的动态采集。研制的样机在微光实验室进行了三基色采集及融合实验,实验时光照度分别1×10-1 lx （等效于月光下）和1×10-3 lx（等效于星光下）,采集速度设置为60 f/s,对获取的图像质量进行了评价。结果表明:在照度为1×10-1 lx时,融合后的真彩色图像在灰度均值、灰度方差和信息熵3项指标方面,比3幅单色图像的平均值分别提高了5.06%、5.97%和1.08%;在照度为1×10-3 lx时,融合后的真彩色图像与3幅单色图像的平均值分别提高了13.18%、-9.86和8.65%。     

荧光寿命显微成像技术及应用的最新研究进展

由于荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响,荧光寿命显微成像技术(fluorescence lifetime imaging microscopy, FLIM)在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点,近年来已被广泛应用于生物医学等领域.然而,尽管FLIM的发明和发展已历经数十年时间,其在实际应用中仍然面临着许多挑战.例如,其成像分辨率受衍射极限限制,而其成像速度与成像质量和寿命测量精度则存在相互制约的关系.近几年来,相关硬件和软件的快速发展及其与其他光学技术的结合,极大地推动了FLIM技术及其应用的新发展.本文简要介绍了基于时域和频域的不同寿命探测方法的FLIM技术的基本原理及特点,在此基础上概述了该技术的最新研究进展,包括其成像性能的提升和在生物医学应用中的研究现状,详细阐述了近几年来研究者们通过硬件和软件算法的改进以及与自适应光学、超分辨成像技术等新型光学技术的结合来提升FLIM的成像速度、寿命测量精度、成像质量和空间分辨率等方面所做的努力,以及FLIM在生物医学基础研究、疾病诊断与治疗、纳米材料的生物医学研究等方面的应用,最后对其未来发展趋势进行了展望.     

Confocal fluorescence lifetime imaging of free calcium in single cells

Ca²⁺ concentrations in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for free calcium and exhibit marked changes in their photophysical properties upon binding. The differences in the fluorescent lifetime of the probes can now be used as a contrast mechanism for imaging purposes. This technique can be further exploited for the quantitative determination of ion concentrations within the cells. We describe the use of a fast fluorescence lifetime imaging method in combination with a standard confocal laser scanning microscope for the determination of Ca²⁺ concentrations in single rat cardiac myocytes using the intensity probe Calcium Green.     

Processing of Fluorescence Lifetime Image Using Modified Phasor Approach: Homo-FRET from the Acceptor

As the hardware of FLIM technique becomes mature, the most important criterion for FLIM application is the correct interpretation of its data. In this research, first of all, a more orthogonal phasor approach, called as Modified Phasor Approach (MPA), is put forward. It is a way to calculate the lifetime of the complex fluorescent process, and a rule to measure how much the fluorescence process deviates from single exponential decay. Secondly, MPA is used to analysis the time-resolved fluorescence processes of the transfected CHO-K1 Cell lines expressing adenosine receptor A₁R tagged by CYP and YFP, measured in the channel of the acceptor. The image of the fluorescence lifetime and the multiplication of the fluorescence lifetime and deviation from single exponential decay reveal the details of the Homo-FRET. In one word, MPA provides the physical meaning in its whole modified phasor space, and broadens the way for the application of the fluorescence lifetime imaging.     

单个心肌细胞内钙波的微观动力学研究

利用基于近场光学原理构建的全内反射荧光显微镜研究了大鼠单个心肌细胞中的钙信号. 利用这种显微镜的快速成像和高信噪比的特点，观察到单个细胞中复杂的二维钙波斑图. 分析了单个钙信号释放事件在钙波形成、运动过程中的作用. 建立在fire-diffuse-fire模型基础上的模拟显示，由基本钙释放事件组成的钙波可以在心肌细胞中稳定存在. 此研究对进一步认识活体可激发系统的微观动力学行为有指导意义. 关键词： 近场光学 全内反射荧光显微镜 心肌细胞 钙波     

Time-lapse in situ fluorescence lifetime imaging of lipid droplets in differentiating 3T3-L1 preadipocytes with Nile Red

To study the mechanisms of and conditions for adipogenesis, an accurate in situ observation tool is necessary to monitor the quantity of intracellular neutral lipids in differentiating preadipocytes. Although conventional fluorescence intensity imaging is a powerful tool for observing the formation and growth of an individual lipid droplet, it suffers from photobleaching and ambiguous autofluorescence or background signals from cells. In this paper, we present a fluorescence lifetime imaging microscopy (FLIM) technique that has the potential to quantify the ratio of neutral to polar lipids in a cell. Measurement of time-lapse FLIM images of differentiating 3T3-L1 cells that contained the Nile Red (NR) probe showed that the average lifetime of NR decreased from 4 ns in preadipocytes to 3 ns in fully differentiated adipocytes after 10 days of differentiation. This large change in the lifetime of NR can be used to monitor the early stages of adipogenesis, even when the lipid droplet is too small to be identified with a conventional microscope.     

门控型像增强器开门时间测量

提出了采用超快激光脉冲与光纤阵列形成的光延时、跟CCD相机相结合的方法,对门控型像增强器进行了开门时间的测量,分析了该测量方法的可行性,建立了门控型像增强器开门时间的测量系统。用该测量方法对超高速光电分幅相机中的门控型像增强器开门时间进行了测量,得到了10,20,30,50 ns档开门时间的实验图片,与所加的快高压脉冲时间12.50,18.50,28.75,48.60 ns相比较,开门时间的测量精度得到了提高,该测量方法可用于超高速光电分幅相机曝光时间的标定。     

神龙一号参数测量用高速两分幅相机

采用长焦距镜头的后工作空间全口径分光原理,利用门控型像增强器、CCD相机、基于大规模可编程集成电路的高速快门控制触发系统等部件,研制了具有较高时间分辨能力和高灵敏度的两分幅高速相机,并在此基础上建立了束参数的高速测量系统。两分幅相机的最高快门速度约3 ns,幅间间隔时间则具有以0.5 ns的步进进行调节的能力;快门时间及幅间间隔时间可以分别独立调节,最大可到1 s;同时具有较好的线性度和空间响应的均匀性,等效背景噪声低到约5 electronspixel-1s-1,并且分幅相机灵敏度调节范围大。该系统一次可以拍摄两幅图像,图像阵列可达到1 0241 024,满足神龙一号的各种测量要求。     

红外激光对可见光成像系统的硬损伤

采用波长为1315nm的连续波化学氧碘激光对某型摄像机进行了辐照实验,研究了可见光CCD成像系统在响应波段外红外激光辐照下的硬损伤效应。开展了光学系统变光圈尺寸下的激光辐照实验,发现当辐照水平一定的情况下,光圈尺寸越小,光学系统越难发生硬损伤,并解释了该现象的成因;测量到光圈尺寸最大、最小两种状态下的光学系统硬损伤功率阈值分别为几十W、几百W;得到CCD的激光损伤阈值为5.5×104W/cm2;结合相机输出的视频图像与显微镜拍到的被损感光器件实物照片分析了硬损伤机理。     

Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochemical properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radio frequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is separated in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calculate in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.