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.     

Design and properties study of fiber optic glucose biosensor

A new type of fiber optic glucose biosensor based on fluorescence quenching has been designed and its properties have been studied. Glucose can be oxidized by oxygen when glucose oxidase are used as the catalyst, therefore, the concentration of glucose can be measured by detecting the consumption of oxygen. For the detection of oxygen concentration, the ruthenium(Ⅱ) complex, Ru(bpy)3Cl2, were used as the fluorescence indicator and its fluorescence lifetime were detected by lock-in technology. The detecting range of the sensor is 50 - 500 mg/dl and its response time is 30 seconds, showing that this kind of sensors is possible to be used in clinical diagnosis and detection.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术，对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究，尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明，利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团，利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具，对眼底细胞随年龄增长的衰老机理的研究具有重要的意义.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术,对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究,尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明,利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团,利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具,对眼底细胞随年龄增长的衰老机理的研究具有重要的意义. 关键词： 双光子激发荧光 荧光寿命成像 视网膜色素上皮层     

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.     

Correction of the quenching effect in two-dimensional laser-induced fluorescence measurement of laser-ablation processes

Laser-induced fluorescence (LIF) and two-dimensional laser-induced fluorescence were used for measurement of the fluorescence lifetime of the A(1)?(+) state of BaO molecules in the laser-ablation process of BaTiO(3) and YBa(2)Cu(3)O(7-x) in the oxygen background gas. The zero-pressure lifetime and the quenching rate were also determined as tau(0)=357ns and k=5.1 x 10(-6)ns(-1)mTorr(-1) , respectively. The spatiotemporal changes of the collisional quenching of BaO molecules were successfully visualized, and the influence of the quenching on the spatial distribution imaging of BaO molecule was corrected.     

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.     

Simultaneous one-dimensional fluorescence lifetime measurements of OH and CO in premixed flames

A method for simultaneous measurements of fluorescence lifetimes of two species along a line is described. The experimental setup is based on picosecond laser pulses from two tunable optical parametric generator/optical parametric amplifier systems together with a streak camera. With an appropriate optical time delay between the two laser pulses, whose wavelengths are tuned to excite two different species, laser-induced fluorescence can be both detected temporally and spatially resolved by the streak camera. Hence, our method enables one-dimensional imaging of fluorescence lifetimes of two species in the same streak camera recording. The concept is demonstrated for fluorescence lifetime measurements of CO and OH in a laminar methane/air flame on a Bunsen-type burner. Measurements were taken in flames with four different equivalence ratios, namely ? = 0.9, 1.0, 1.15, and 1.25. The measured one-dimensional lifetime profiles generally agree well with lifetimes calculated from quenching cross sections found in the literature and quencher concentrations predicted by the GRI 3.0 mechanism. For OH, there is a systematic deviation of approximately 30 % between calculated and measured lifetimes. It is found that this is mainly due to the adiabatic assumption regarding the flame and uncertainty in H₂O quenching cross section. This emphasizes the strength of measuring the quenching rates rather than relying on models. The measurement concept might be useful for single-shot measurements of fluorescence lifetimes of several species pairs of vital importance in combustion processes, hence allowing fluorescence signals to be corrected for quenching and ultimately yield quantitative concentration profiles.     

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。     

凝聚相有机分子的激发能传递研究(Ⅱ)——激光染料二甲基-POPOP和DCM分子间的激发能传递

利用荧光发射及其衰变动力学测量,研究了激光染料分子二甲基-POPOP和DCM在溶液中的分子间能量传递。所得结果证实:激发态二甲基-POPOP分子荧光被猝灭的程度,随所加入DCM浓度的增大而增大。尽管DCM的荧光寿命随二甲基-POPOP的猝灭而增加。然而,DCM的加入并不改变二甲基-POPOP的荧光寿命。在固定DCM浓度,而增加二甲基-POPOP浓度时,相似的结果同样被观测到。这些结果表明:激发态二甲基-POPOP分子通过辐射传能机理向基态DCM传递激发能,其结果将入射光的波长从紫外直接转换到波长大于600nm的光谱区。在本文中也简要讨论了进一步改善这一传能过程效率的措施。     

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.     

Transient absorption spectroscopy of a monofullerene C<Subscript>60</Subscript>-bis-(pyropheophorbide <Emphasis Type="Italic">a</Emphasis>) molecular system in polar and nonpolar environments

The population dynamics of the excited and ground states of the monofullerene-bis (pyropheophorbide a) complex (FP1) were studied in polar (DMF) and nonpolar (toluene) solvents using picosecond transient absorption techniques. A strong quenching of the fluorescence signal of FP1 was observed in both solvents, in comparison to the fluorescence of bis (pyropheophorbide a) (P2). This quenching is due to an intramolecular photoinduced electron transfer from the pyropheophorbide a (pyroPheo) moiety to the fullerene C₆₀ monoadduct. In DMF the charge-separated (CS) state of FP1 has a lifetime of 0.32 ns and undergoes a direct transition to the ground state, resulting in a very low value of photosensitised singlet oxygen generation. In toluene, energy transfer from the first excited triplet state of pyroPheo, which has been populated via relaxation of the CS state, generates a considerable amount of singlet oxygen. The lifetime of the CS state in the nonpolar solvent was estimated to be 0.29 ns. It was also shown that in both DMF and toluene the first excited singlet state as well as the triplet state of the fullerene moiety in FP1 are not occupied. PACS 31.70.Dk; 31.70.Hq; 33.50.-j; 34.70.+e     

Lifetime measurements,infrared and photoacoustic spectroscopy of NdP5O14

We have made a series of spectroscopic measurements to better understand the optical properties and concentration quenching characteristics of the stoichiometric laser material NdP₅O₁₄. The results of fluorescence lifetime measurements in the presence of different surface environments indicates that the surface condition affects the concentration quenching whereas lifetime measurements under applied uniaxial stress show that the presence of internal strains in the crystal is not significantly effective in fluorescence quenching. A comparison of the internal reflection spectrum with the normal infrared spectrum did not reveal any significant differences in the Nd³⁺ energy levels. Photoacoustic spectroscopy results proved difficult to interpret but appear to be consistent with the presence of surface quenching of excitons and indicate that the intrinsic quantum efficiency of Nd³⁺ ions in the pentaphosphate host in the absence of concentration quenching is approximately 0.90.     

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.     

醌对卟啉荧光猝灭的溶剂效应

测定了在室温和未脱氧条件下不同浓度的苯醌(BQ)对卟啉衍生物四苯基卟吩(TPP),四苯基卟吩锌(ZnTPP)激发一重态的猝灭.由猝灭而引起的荧光强度的变化和荧光寿命的变化均可用Stern-Volmer关系式描述.由此根据寿命计算出的猝灭常数均比据荧光强度计算出的猝灭常数小.这是由于荧光强度测量中的自吸收效应引起的.实验结果表明;猝灭常数Kq与溶剂极性无关而只与粘度有关.说明这种猝灭是受扩散控制的动态猝灭.动力学分析表明,这是一种强猝灭,电荷转移猝灭速度比卟啉和醌的复合物的分解速度大或与之相近.比较实验结果和我们以前及其他作者发表的结果后,可以得出结论.苯醌对卟啉衍生物激发态电荷转移猝灭的溶剂效应随分子状态的不同而异:分子内的和通过三重态进行的分子间电荷转移猝灭受溶剂极性影响,但通过一重态进行的分子间电荷转移猝灭则与溶剂极性无关.     

基于平行因子分析的激光诱导纳秒时间分辨荧光猝灭法原位研究芘和菲与腐植酸相互作用

基于平行因子(PARAFAC)分析的激光诱导纳秒时间分辨荧光(LITRF)猝灭法原位研究典型多环芳烃(PAHs)芘(Pyr)和菲(Phe)单独及混合状态下与Aldrich腐植酸(HA)相互作用。实验表明,在266 nm激发波长下,Pyr,Phe及HA三者的LITRF光谱相互重叠,无法直接同时测定混合组分中游离Pyr和Phe荧光强度。利用PARAFAC分析可快速有效消除HA荧光干扰,获取混合溶液LITRF光谱中Pyr和Phe荧光强度及各自荧光衰减曲线,并以Freundlich吸附等温模型描述PAHs与HA结合特性。结果验证了Pyr和Phe与HA以非线性形式结合(n<1);Pyr和Phe共存时,两者与HA结合存在竞争关系。加入竞争吸附质后,Pyr和Phe与HA的吸附等温线非线性程度减弱(n趋近于1),单点结合系数下降,且竞争强度随竞争吸附质浓度增加而增强。此外,LITFR-PARAFAC猝灭法与传统荧光猝灭法所得单组分Pyr和Phe与HA结合特性无显著差异。猝灭速率常数及荧光寿命分析反映出Pyr和Phe与HA间荧光以静态猝灭形式为主。LITFR-PARAFAC猝灭法可快速原位研究混合PAHs与HA相互作用,有利于原位预测和评估PAHs的环境行为及其生态风险。     

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。     

Analysis of fluorescence quenching of pyronin B and pyronin Y by molecular oxygen in aqueous solution

The fluorescence quenching of pyronin B and pyronin Y molecules by molecular oxygen in aqueous solution was studied by using steady-state and time-resolved fluorescence and UV-Vis absorption spectroscopy techniques. In order to understand the quenching mechanism, fluorescence decays, absorption and fluorescence spectra of the probes were recorded as a function of the oxygen concentration and temperature. The quenching was found to be appreciable and shows positive deviation in the Stern-Volmer representation obtained from the fluorescence intensity ratio. Fluorescence quenching constants (k_q) were calculated from the τ_o/τ vs. [Q] plots having linear correlation and compared with calculated diffusion-controlled rate constants (k_diff) values. Experimental results were in good agreement with the simultaneous dynamic and static quenching model.     

Validation of the protoporphyrin IX-triplet state lifetime technique for mitochondrial oxygen measurements in the skin

Mitochondrial oxygen tension can be measured in vivo by means of oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Here we demonstrate that mitochondrial PO(2) (mitoPO(2)) can be measured in the skin of a rat after topical application of the PpIX precursor 5-aminolevulinic acid (ALA). Calibration of mitoPO(2) measurements was done by comparison with simultaneous measurements of the cutaneous microvascular PO(2) This was done under three different conditions: in normal skin tissue, in nonrespiration skin tissue due to the application of cyanide, and in anoxic skin tissue after the ventilation with 100% nitrogen. The results of this study show that it is feasible to measure the mitoPO(2) after the topical application of ALA cream by means of the PpIX-triplet state lifetime technique.     

Luminescence and anisotropy decays ofN-3-pyrene maleimide labeling IgG proteins and cells

The Py.M (N-3-Pyrene Maleimide) is a dye that covalently binds to reactive amino or sulfhycryl groups to give highly fluorescent protein conjugates. Measurements of luminescence lifetimes and anisotropy decays have been performed with a Phase and Modulation Fluorometer. Complexes of Py.M-antibody (IgG antimouse) and tumoral cells C6 labeled with Py.M have been investigated. The Py.M fluorescence in buffer solution and the protein and cells natural fluorescence have been checked. For Py.M-IgG and labeled cells, the fluorescence decays present interesting behaviours. The least-squares analysis of the experimental results on Py.M-IgG complex points out two lorentzian distributions centered at 74 ns and 11 ns, on the contrary, for the labeled cells, a discrete component at 100 ns and a lorentzian distribution centered at 5 ns are shown. In both systems a weak component lower than 1 ns is observed. The fluorescence decays, mainly the long lifetime one, are very sensitive to oxygen quenching, showing the high efficiency of O₂ quenching. For samples N₂ bubbled, the lifetime experimental resuits show a decrease of the oxygen accessibility from free probe in solution to Py.M-IgG complex and to labeled cells, compatible with a more compact packing of the probe binding site. The experimental results of anisotropy decays of degassed samples show for Py.M-IgG complexes a long rotation correlation time of about 200 ns at T=5°C, assigned to overall rotation of the protein, besides shorter correlation times attributable to inner protein motions. For labeled cells, the long rotation correlation time becomes of the order of 580 ns confirming a progressive increase of the stabilization of the binding site.