Global Analysis of Dynamic Fluorescence Anisotropy by a Polarized Phasor Approach

Recently, the graphical analysis of the fluorescence lifetime imaging using the phasor approach has been highlight, and a series of the reports have made it on the way for the applications by the nonprofessionals. In this paper, we put forward a similar theory validated by the experiments for the dynamic fluorescence anisotropy imaging. By subtracting the perpendicular component from the parallel one in the frequency-domain polarization measurement, we deduce a new analytical expression about the fluorescence joint time, and find that as much as the fluorophore is a single exponential decay and $ {r_\infty } $ is equal to zero, ??I(t) is a single exponential decay with the time constant X as well, and the center of its histograms is located on the semicircle in the polarized phasor plot. In the end, we conclude that the fluorescence joint time is the best parameter to weigh the fluorescence dynamics for the macromolecules.     

The Effect of Translational Motion on FLIM Measurements-Single Particle Phasor-FLIM

Fluorescence lifetime imaging microscopy or FLIM provides a versatile tool for spatially-mapping macromolecular interactions and environments through pixel-by-pixel resolution of the excited-state lifetime. In conventional frequency-domain FLIM the phase and modulation of the detected fluorescence are determined by the photophysics of the fluorophore only. However, translational motion on the timescale of FLIM acquisition can significantly perturb apparent phase and modulation values owing to intensity fluctuations and phase decoherence. Using the phasor plot we outline a simple analytic theory, numerical simulations and measurements on fluorescent beads (ex 470 nm, em 520 nm). Fluctuations due to particle motions result in an increase in the number and spread of phasors, an effect we refer to as phasor broadening. The approach paves the way for the measurement of lifetimes and translational motion from one experiment.     

拟威布尔分布密度函数在荧光寿命成像数据分析中的应用

荧光寿命法成像技术(FLIM)是一种非常有效、功能强大且能用来分析复杂生物组织和细胞分子的成像技术。传统的荧光寿命成像的数据分析,按某些具有不同寿命、离散的单参量指数模型来描述荧光衰减过程。在生物组织这样既复杂又不均匀的样品中,虽然多参量指数模型能提供比单参量指数模型对实验数据更好的拟合效果,但是离散多参量的假定往往是随意的。提出了拟威布尔分布密度函数可能是生物荧光分子团衰减动力过程的真实再现,并且通过计算证明,对于某些生化感兴趣的荧光分子团的多槽基面效价测定样品的数据,相对于单参量指数与多参量指数衰减函数有更好的一致性。同时讨论了将该荧光衰减模型应用于荧光寿命成像的前景。     

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.     

DMANS——一种新型荧光闪烁体染料的寿命与能量转移的研究

本工作对一种新型荧光闪烁体染料——4-二甲胺基—4''硝基芪(DMANS)在不同介质中的光谱行为和能量转移进行了研究,发现介质的极性大小以及介质和染料间的能量转移对该闪烁体染料的荧光量子产率和闪烁发光延迟具有重大影响。研究对选择基体材料和合理组成闪烁材料配方有一定的参考价值。     

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     

单光子调制频谱用于量子点荧光寿命动力学的研究

本文开展了基于单光子调制频谱测量量子点荧光寿命动力学特性的研究.在脉冲激光激发下,对探测到的量子点单光子荧光信号进行频谱分析以获得荧光调制频谱,研究发现特征频谱信号幅值与荧光寿命之间存在确定的非线性对应关系.这种单光子调制频谱方法能有效消除背景噪声和单光子探测器暗计数的影响,用于分析量子点荧光寿命动力学特性时在准确度以及时间分辨率方面都较目前普遍采用的荧光衰减曲线寿命拟合方法呈现出明显优势:当涨落误差为5%时,寿命测量准确度提高了一个数量级;当涨落误差和偏离误差均为5%时,对动力学测量效率以及时间分辨率提高了四倍以上.因此单光子调制频谱可以作为获取量子点在短时间尺度内激发态动力学信息的一种有效技术手段.     

Optimum pulse repetition rates for single photon counting experiments

In the first part of this paper, the optimum pulse repetition rate (PRR) for fluorescence lifetime measurements based on time correlated single photon counting is obtained by minimizing the variance in the lifetime estimates, assuming a single exponential decay. This allow us, in the second part, to calculate the optimum pulse repetition rate for systems possessing a set of discrete repetition rates; such as synchronously pumped mode locked cavity dumped laser systems.     

Time-resolved OSL studies of YAlO3:Mn2+ crystals

Time-Resolved Optically Stimulated Luminescence (TR-OSL) from single crystalline YAlO₃:Mn²⁺ samples was investigated using a green light emitting diode (λ ∼ 525 nm) as stimulation light source. The TR-OSL decay curve of the material can be described with a single exponential decay function with a lifetime about 80 ms that does not depend on irradiation dose in the range from 50 mGy to 1 kGy. This OSL decay is superposed on a photoluminescence signal with a much shorter (3.5 ms) decay lifetime. The Mn²⁺ photoluminescence decay with a lifetime of 3.5 ms can be easily eliminated by corresponding time resolution using pulsed OSL readout. Dose response and thermal stability of the OSL signal are consistent with the previous thermoluminescent (TL) studies of the material.     

Time-resolved photoluminescence study of Ga In P alloys

The time-resolved photoluminescence spectra of ordered and disordered Ga_0.52In_0.48P alloys were studied at room temperature and at 77 K liquid nitrogen, respectively. The ordered samples have well fitted two exponential processes decay curves and the time constants are sample dependent and have little relationship with the ordering degree. The decay curve of disordered sample shows that it has single exponential process and its lifetime has a tendency of reduction with the decrease of excitation intensity. The photoluminescence spectra with different delay time at 77 K show that the ordered samples exhibit about 6 ∼ 10 meV blue-shift of PL peak energy with the delay time. Received 13 December 2001 Published online 19 July 2002     

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.     

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.     

Luminescence of biologically active 24-epicastasterone and a model compound

Biologically active brassinosteroid 24-epicastasterone, ring B of which contains a C=O group and has the nπ*-configuration for a low-lying electronic excited state, exhibits rapid fluorescence. The wavelengths of the fluorescence maxima of the steroid dissolved in hexane and acetonitrile are equal to 332 and 394 nm, respectively. The fluorescence lifetime of the steroid dissolved in acetonitrile is τ = 9.9 nsec. Solutions of 24-epibrassinolide do not luminesce. The long-wavelength electronic absorption band λ_max^abs = 340 nm in the absorption spectrum of an ethanol solution of model compound 2, ring D of which contains a C=O group π*-conjugated with the C=C double bond of ring C, like in the spectrum of the steroid, has a low extinction coefficient. An ethanol solution of 2 does not fluoresce. 24-Epicastasterone at 77 K in ethanol solution exhibits phosphorescence with λ_max^phos = 447 nm. The phosphorescence decay is exponential with τ = 0.79 msec. Compound 2 also phosphoresces. The phosphorescence spectrum of its ethanol solution has a maximum at 490 nm. The phosphorescence decay is nonexponential in the early stage. The phosphorescence lifetime is 25 msec in the exponential decay region. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 2, pp. 182–186, March–April, 2008.     

Maximum Entropy Analysis of Analytically Simulated Complex Fluorescence Decays

We tested a Maximum Entropy Method developed for oversampled data (SVD-MEM) on complex analytically simulated exponential decay data consisting of both noisy and noiseless multi-exponential fluorescence decay curves. We observed recovery of simulated parameters for three sets of data: a decay containing three exponential functions in both intensity and anisotropy curves, a set of intensity decays composed of 4, 5 and 6 exponential functions, and a decay characterized by a Gaussian lifetime distribution. The SVD-MEM fitting of the noiseless data returned the simulated parameters with the high accuracy. Noise added to the data affected recovery of the parameters in dependence on a data complexity. At selected realistic noise levels we obtained a good recovery of simulated parameters for all tested data sets. Decay parameters recovered from decays containing discrete lifetime components were almost independent of the value of the entropy scaling parameter γ used in the maximization procedure when it changed across the main peak of its posterior probability. A correct recovery of the Gaussian shaped lifetime distribution required selection of the γ-factor which was by several orders of magnitude larger than its most probable value to avoid a band splitting.     

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.     

The time evolution of compound elastic scattering measured by crystal blocking

Using the crystal blocking technique, we have measured the emission distributions of protons near the 〈110〉 axis elastically scattered from a 7000 Å thick single nickel crystal, at 5.65 and 6.50 MeV. These distributions are fitted by an analytic multiple string calculation of the blocking process which yields the time-dependent compound-nucleus decay probability curve. Shape- and compound-elastic cross sections are simultaneously determined in a reaction-model-independent way, as is the mean compound nucleus lifetime. The resulting decay curves for the delayed component are consistent with a single exponential at both energies and show no indication of the presence of level-level correlations. Use of such data in testing recent statistical theories is discussed.     

Photobleaching-Based Quantitative Analysis of Fluorescence Resonance Energy Transfer inside Single Living Cell

The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensity-based FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH ₁ ) to selectively detect the fluorescence from donor, and a FRET channel (CH ₂ ) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb-FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring protein-protein interactions inside single living cell.     

CVD grown 2D MoS2 layers: A photoluminescence and fluorescence lifetime imaging study

In this letter, we report on the fluorescence lifetime imaging and accompanying photoluminescence properties of a chemical vapour deposition (CVD) grown atomically thin material, MoS₂. µ‐Raman, µ‐photoluminescence (PL) and fluorescence lifetime imaging microscopy (FLIM) are utilized to probe the fluorescence lifetime and photoluminescence properties of individual flakes of MoS₂ films. Usage of these three techniques allows identification of the grown layers, grain boundaries, structural defects and their relative effects on the PL and fluorescence lifetime spectra. Our investigation on individual monolayer flakes reveals a clear increase of the fluorescence lifetime from 0.3 ns to 0.45 ns at the edges with respect to interior region. On the other hand, investigation of the film layer reveals quenching of PL intensity and lifetime at the grain boundaries. These results could be important for applications where the activity of edges is important such as in photocatalytic water splitting. Finally, it has been demonstrated that PL mapping and FLIM are viable techniques for the investigation of the grain‐boundaries.

     

Exciton emission dynamics in single InAs/GaAs quantum dots due to the existence of plasmon-field-induced metastable states in the wetting layer

A very long lifetime exciton emission with non-single exponential decay characteristics has been reported for single InA-s/GaAs quantum dot(QD) samples,in which there exists a long-lived metastable state in the wetting layer(WL)through radiative field coupling between the exciton emissions in the WL and the dipole field of metal islands.In this article we have proposed a new three-level model to simulate the exciton emission decay curve.In this model,assuming that the excitons in a metastable state will diffuse and be trapped by QDs,and then emit fluorescence in QDs,a stretchedlike exponential decay formula is derived as I(t)=At~(β-1)e~(-(rt)β),which can describe well the long lifetime decay curve with an analytical expression of average lifetime     

Photophysical Studies on the Interaction of Acridinedione Dyes with Universal Protein Denaturant: Guanidine Hydrochloride

Photophysical studies of photoinduced electron transfer (PET) and non-PET based acridinedione dyes with guanidine hydrochloride (GuHCl) were carried out in water and methanol. Addition of GuHCl to photoinduced electron transfer (PET) based acridinedione dye (ADR 1) results in a fluorescence enhancement, whereas a non-PET based dye (ADR 2) shows no significant change in the fluorescence intensity and lifetime. Addition of GuHCl to ADR 1 dye in methanol results in single exponential decay behaviour, on the contrary a biexponential decay pattern was observed on the addition of GuHCl in water. Absorption and emission spectral studies of ADR 1 dye interaction with GuHCl reveals that the dye molecule is not in the protonated form in aqueous GuHCl solution, and the dye is confined to two distinguishable microenvironment in the aqueous phase. A large variation in the microenvironment around the dye molecule is created on the addition of GuHCl and this was ascertained by time-resolved area normalized emission spectroscopy (TRANES) and time-resolved emission spectroscopy (TRES). The dye molecule prefers to reside in the hydrophobic microenvironment, rather in the hydrophilic aqueous phase is well emphasized by time-resolved fluorescence lifetime studies. The mechanism of fluorescence enhancement of ADR 1 dye by GuHCl is attributed to the suppression of the PET process occuring through space.