Fluorescence Recovery Under Decaying Photobleaching Irradiation: Concept and Experiment

A novel modification of photobleaching method for measurement of lateral diffusion is developed. In this approach fluorescence recovery kinetics is measured under decaying photobleaching irradiation, termed as fluorescence recovery under decaying photobleaching (FRDP). The time evolution of fluorescence intensity normalized to input irradiation starts from the photobleaching kinetics and transforms into the kinetics of fluorescence recovery at a later stage resulting in appearance of minimum. The analytical solution for the kinetics of fluorescence for Gaussian lineshape of laser beam and hyperbolic decay of irradiation in the first order approximation on bleaching rate was obtained. The accuracy of the analytical function was evaluated with exact numerical solution computed with finite differentiates method. The FRDP method was successfully applied to fluorescein solution in the glycerol/water mixture (80%) under various experimental settings using home-made experimental set-up. The FRDP approach demonstrated 25–30 fold enhancement in signal intensity over classical fluorescence recovery after photobleaching (FRAP) method at 3–5 fold increase in total irradiation. Among other advantages of the FRDP is the opportunity to perform measurements on varying time scales under constant size of the bleaching spot, including “safe” long time measurements. The potential extra advantage of FRDP method for analysis of complex diffusion in the biological system is discussed.     

荧光光谱用于光动力疗法中光敏剂光漂白特性研究

应用荧光光谱技术研究溶液中血卟啉单甲醚(HMME)的光漂白与光产物生成。以532 nm倍频Nd∶YAG激光器照射样品,功率密度为100 mW·cm-2,以光学多通道分析仪(OMA)采集荧光光谱。照光过程与荧光光谱采集同步进行。通过构建基本光谱与最小二乘拟合,由单条实测光谱中分解求得HMME荧光(613 nm)、光产物荧光(639 nm)及自体荧光的强度。HMME初始浓度不超过10 μg·mL-1时符合荧光-浓度线性函数关系。对照光过程的荧光光谱监测同时观察到HMME漂白、光产物生成与漂白,以及样品光学特性变化引起的自体荧光强度起伏。光产物漂白后的二次产物引起样品光学特性显著改变。所建立的荧光光谱探测系统与光谱分析方法可满足光敏剂漂白特性体外研究的需要,并为光动力治疗的剂量学在体监测提供有效研究方法。     

厦门湾有色溶解有机物光漂白的三维荧光光谱研究

利用荧光激发-发射矩阵光谱(excitation-emission matrix spectroscopy,EEMs)研究了厦门湾九龙江河口中、低盐度区表层水样中有色溶解有机物在秋季天然太阳辐照下的光化学漂白。结果表明,光漂白未引起水样中类腐殖质(C, A, M)和类蛋白质(T,B)荧光峰位置的移动。5个峰的荧光强度均随辐照时间的增加而减少,其中低盐度水样的降低幅度更大,并以指示陆源河流输入的特征荧光峰C的光漂白程度最大。根据漂白速率可将各荧光团区分出易漂白和难漂白两类组分。光漂白导致T、C峰及A、C峰之间的荧光强度比值增加,说明光漂白可引起海水中溶解有机物性质的明显改变,并且也是近海类蛋白质荧光相对类腐殖质荧光占优势的一个重要控制因素。研究结果对于探究陆源有机物在近海的转化及去除过程以及海洋光学有一定的参考价值。     

基于明胶制备碳量子点及其光学性能的研究

通过水热法采用热解明胶制备出有蓝色荧光的碳量子点,并通过单因素优化实验对制备碳量子点的温度、时间进行优化以选择出制备碳量子点的最佳条件,结果表明在水热反应温度为200 ℃,反应时间为6 h时制备的碳量子点的荧光性能最强。同时,利用透射电子显微镜(TEM)、傅里叶变换红外光谱(FTIR)、X射线光电子能谱(XPS)、Ｘ射线衍射(XRD)、紫外-可见吸收光谱(UV)及荧光光谱(PL)等手段对最佳条件下制备的碳量子点进行测试与表征,结果表明,该方法制备的碳量子点量子产率为39.4%,与不掺杂的碳量子点相比其量子产率相对较高,这可能是因为有N元素的存在使得量子产率有所提高;所制备的碳量子点不仅具有丰富的含氧官能团而且抗光漂白性能良好,形态主要是均匀分散的球形,没有明显的晶格条纹,这与相关文献报道的碳量子点的形态相一致,其在250～300 nm有较弱的吸收,但无明显的特征吸收峰,这可能是由于C=O基团的n-π*跃迁引起的;此外,还讨论了氙灯照射时间、pH、碳量子点浓度、不同类型溶剂及离子强度等因素对碳量子点荧光性能的影响,研究结果表明,氙灯照射时间及离子强度对碳量子点荧光性能几乎无影响,在过酸或过碱的条件下其荧光强度相对较弱,原因可能是在过酸或过碱的条件下发生质子化或非质子化的作用导致其荧光强度减弱;且碳量子点溶液随着其浓度的增加,荧光强度先增加后减小;而对于溶剂类型而言,其在极性溶剂中的荧光强度大于其在非极性溶剂中的荧光强度,说明该方法制备的碳量子点具有良好的水溶性。     

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.     

Photobleaching with a subnanosecond laser flash

In standard fluorescence recovery after photobleaching (FRAP) applications for measuring lateral diffusion rates and adsorption/desorption kinetics of fluorescent molecules at biological or model membranes, irreversible bleaching is induced by a bright excitation flash of at least millisecond time scale. It has been presumed that the bleaching event is of a low probability and the significant bleached population that develops during the flash results from each molecule undergoing thousands of excitation/deexcitation cycles before a bleaching event occurs. In some FRAP experiments, notably polarized FRAP (PFRAP) for measuring molecular rotational diffusion rates, it is desirable to use much shorter (subnanosecond) bleaching pulses. However, subnanosecond pulses are shorter than the fluorescence lifetime, so that any fluorophore will experience at most only one visit to the excited state during the bleaching pulse. If bleaching occurs only by the same processes as in slower FRAP experiments, one would thereby expect only minimal bleaching regardless of the bleach intensity. Moreover, the ability of fast polarized pulses to imprint an anisotropic orientational pattern in the postbleach unbleached fluorophore, an ability essential for PFRAP, is not at all guaranteed, particularly if two-photon processes are involved in high-intensity short bleach pulses. In this study, bleaching depths are measured as a function of subnanosecond pulse intensity on a small labeled protein covalently immobilized on fused silica. We show that bright subnanosecond laser flashes do indeed produce significant bleaching, that both two photon effects and reversible bleaching are involved, and that polarized bleaching does produce an anisotropic orientational pattern of unbleached fluorophore. We also postulate a theoretical molecular state model which semiquantitatively accounts for the experimentally observed dependence of reversible bleaching on bleaching pulse intensity.     

Correlation of cytotoxicity and depth of necrosis of the photoproducts of photogem®

Photodynamic therapy (PDT) is an approved modality for cancer treatment, which involves the administration of a photosensitive drug (PS) that is selectively accumulated in neoplastic tissues and their vasculature and subsequently can be activated with light at the appropriate wavelength to generate reactive molecular species that are toxic to tissues. In PDT, a great part of the used PS suffers degradation by light (photobleaching) that involves a decrease in the absorption and intensity of fluorescence of the photosensitizer as well as photoproduct formation evidenced by the appearance of a new absorption band. In this study, we investigated the correlation of cytotoxicity and depth of necrosis of Photogem and its photoproducts obtained previously by irradiation at 514 and 630 nm. The cytotoxicity for degraded Photogem decreases with the previous irradiation time of Photogem solution suggesting that the photoproducts of Photogem are less cytotoxics than the original formulation. A transition between the necrosed epithelium and healthy epithelium of normal liver of rats after irradiation at 630 nm was observed with irradiated and nonirradiated PS. It is observed that the depth of necrosis only at irradiation dose of 150 J/cm² in both concentrations is greater for Photogem followed by Photogem degradated previously at 514 and then at 630 nm. The results obtained suggest that the threshold of necrosis values is lower for Photogem followed by its photoproducts formed, suggesting that the photoproducts present a low photodynamic activity. If the photosensitizer degradation happens at the same time as tumor destruction, the drug degradation can be complete before reaching the threshold of necrosis; then it is very important to control the drug concentration and light intensity of irradiation during PDT.     

Two-Photon Activated Two-Photon Fluorescence and Binding of Azidocoumarin in a Gelatin Matrix

We study the creation of fluorescence patterns inside a gelatin gel by way of two-photon photoactivation of 7-azido-4-trifluoromethyl-1,2-benzopyrone (azidocomarin 151) contained in the gel matrix. As ultrafast light pulses are focused into the gel, onset of two-photon fluorescence, highly nonlinear in the applied optical power, is observed as azidocoumarin is converted into a fluorescent dye that binds to the gelatin. We fit the time dependence of the fluorescence to a model that incorporates the competition between coumarin photoactivation and photobleaching as well as the gradual degradation of the gel when it is exposed to the high intensity laser light. The model predicts that the initial rate of fluorescence onset should scale as the P (4), where P is laser power, while the signal at long exposure time should scale as P (3/2). The observed exponents are 4.18 and 1.34, respectively. The model allows us to estimate the cross section and quantum yield of two-photon induced photobleaching of azidocoumarin 151. The numerous technical uses of gelatin and the collagen from which it derives in areas ranging from photography to tissue engineering provide possible applications for the techniques described in this paper.     

Photostability comparison of CdTe and CdSe/CdS/ZnS quantum dots in living cells under single and two-photon excitations

The photostability is an outstanding feature of quantum dots (QDs) used as fluorescence probes in biological staining and cell imaging. To find out the related factors in the QD photostability, the photobleaching of naked CdTe QDs and BSA coated CdSe/CdS/ZnS QDs in human hepatocellular carcinoma (QGY) cells and human nasopharynx carcinoma (KB) cells were studied under single photon excitation (SPE) and two-photon excitation (TPE). In these two cell lines the cellular QDs were irradiated by a 405 nm continuous wave laser for SPE or an 800 nm femto-second (fs) laser for TPE. The QD photobleaching with the irradiation time was found to fit a biexponential decay. The fast decay plays a dominant role in the bleaching course and thus can be used as the parameter to quantitatively evaluate the QD photostability. The TPE decreased the QD photobleaching as compared to SPE. The BSA coated core/shell QDs had improved the photostability up to 4-5 times than the naked QDs due to the shielding effect of the QD shell. Therefore, it is better to use core/shell structured QDs as the fluorescence probe combining with a TPE manner for those long-term monitoring studies.     

Segmented Frequency-domain Fluorescence Lifetime Measurements: Minimizing the Effects of Photobleaching Within a Multi-component System

This study introduces a newly developed frequency segmentation and recombination method for frequency-domain fluorescence lifetime measurements to address the effects of changing fractional contributions over time and minimize the effects of photobleaching within multi-component systems. Frequency segmentation and recombination experiments were evaluated using a two component system consisting of fluorescein and rhodamine B. Comparison of experimental data collected in traditional and segmented fashion with simulated data, generated using different changing fractional contributions, demonstrated the validity of the technique. Frequency segmentation and recombination was also applied to a more complex system consisting of pyrene with Suwannee River fulvic acid reference and was shown to improve recovered lifetimes and fractional intensity contributions. It was observed that photobleaching in both systems led to errors in recovered lifetimes which can complicate the interpretation of lifetime results. Results showed clear evidence that the frequency segmentation and recombination method reduced errors resulting from a changing fractional contribution in a multi-component system, and allowed photobleaching issues to be addressed by commercially available instrumentation. Electronic supplementary material The online version of this article doi: contains supplementary material, which is available to authorized users.     

Application of the Doehlert Design to Optimize the Signal Obtained in Photochemically Induced Fluorescence for the Determination of Eight Phenylureas

This work describes the optimization of a photochemically induced method for the detection of eight phenylureas has been developed by response surface methodology (RSM). These pesticides do not show native fluorescence but they were photolyzed into strongly fluorescent photoproducts under UV irradiation. The effect of the main variables affecting the yield of the photoderivatization reaction, and hence the fluorescence intensity, such as solvent, UV irradiation time and pH were optimized for each pesticide. A Doehlert design was applied in order to obtain maximum intensity fluorescence using response surface methodology. In general, a maximum was found for all pesticides using MeOH as organic solvent, except for diuron, whereas the effect of pH and irradiation time was different, according to each pesticide. Finally, the addition of β-cyclodextrin upon the photochemically induced fluorescence intensity was investigate. The fluorescence intensity was only improved for monolinuron at a concentration of 4 × 10⁻³ M of β-cyclodextrin.     

微阵列芯片的荧光光漂白特性

微阵列技术为大量基因表达水平的同时监控提供了一种高效的手段。随着微阵列芯片朝着小型化、高通量和弱信号方向发展,荧光检测技术以其易寻址和高灵敏度等优势越来越受到世人关注。在微阵列技术中,人们通过检测微阵列芯片上不同位置斑点的荧光信号强度,可以得知微阵列芯片不同位置固定的已知序列探针与荧光染料标记的cDNA样品的杂交情况。对一张微阵列芯片多次扫描后,荧光染料发生光漂白,荧光强度发生衰减变化,它将为微阵列的数据分析带来误差。使用荧光浓度梯度微阵列芯片研究了芯片经多次扫描后荧光斑点强度的衰减情况,通过拟合相同荧光斑点经多次重复扫描后得到的信号强度,得到了荧光斑点强度按指数形式衰减的规律,并在此基础上研究了荧光斑点强度衰减指数模型中的参数与荧光斑点初始浓度的关系,为进行微阵列芯片数据光漂白误差修正提供了实验依据。     

A General Method for Constrained Analysis of Fluorescence Anisotropy Decay: Application of the Steady-State Anisotropy

Time-resolved fluorescence anisotropy is an invaluable method for investigating the internal and rotational dynamics of biomolecules. The range of rotational motions detectable by anisotropy decay is limited by the fluorescence lifetime; typically, a depolarizing motion may be resolved if the associated correlation time is between 0.1 and 10 times the intensity decay lifetime. To extend that range and to improve the recovery of anisotropy decay parameters, a general analytical method has been developed. This procedure utilizes a modification of Lagrange multiplier methods to constrain the values of the iterated kinetic parameters during nonlinear least-squares analysis of anisotropy decay data. The form of the constraint equation is derived from the classic relationship between the decay parameters and the steady-state anisotropy, which can be simply and accurately measured. Application of the constraint to analyses of synthetic data sets increased the accuracy of recovery by decreasing the uncertainty in the iterated parameters. The constraint also enabled the accurate recovery of correlation times that were a factor of 30 greater than the fluorescence lifetime, although it did not improve recovery of correlation times that were much shorter than the lifetime. Using this technique, it should now be possible to characterize the dynamics of larger macromolecules and assemblies than those that can currently be studied by fluorescence anisotropy decay.     

氢化物-原子荧光光谱法检测生活饮用水中的痕量铅

建立一种简便准确的氢化物-原子荧光光谱法检测生活饮用水中痕量铅的方法.在酸性介质中,以铁氰化钾为氧化剂,2%的盐酸为载流,样品中的铅与硼氢化钠(NaBH<,4>)或硼氢化钾(KBH<,4>)反应生成铅的挥发性氢化物(PbH<,4>),将氢化物导入原子化器中,检测其荧光强度.铅在0.00-30.0μg/L范围内,荧光强度...     

生物相容性量子点的表征及其在细胞标记中的应用

利用无机低温水相合成法制备表面包覆L-半胱氨酸的CdTe/ZnTe核壳型量子点,测量不同溶液以及激发功率激光作用下该量子点的光谱特性。结果表明,该量子点吸收谱和荧光光谱峰值位置不随pH值变化,而荧光光强随pH值升高呈近似线性上升趋势;不同缓冲液对该量子点荧光光强无影响,但随孵育时间延长,荧光强度略有下降;在强激光照射下QDs会被快速光漂白,选择适当的激光功率(<100 μW),可降低漂白速率,实现长时间稳定测量。因此,该量子点具有较好的生物稳定性和光稳定性,将其与血铁蛋白相连形成靶向共轭纳米粒,可成功用于HeLa细胞标记。细胞内量子点光漂白实验表明,细胞微环境会影响量子点的光稳定性,加速量子点的光漂白。     

An Ensemble and Single-molecule Fluorescence Spectroscopy Investigation of Calcium Green 1, a Calcium-ion Sensor

The calcium-ion indicator dye, Calcium Green 1 (CG-1), has been characterized using a combination of ensemble and single-molecule optical spectroscopy measurements. In terms of ensemble measurements, CG-1 demonstrated a strong increase in fluorescence emission as a function of increasing [Ca²⁺]. This was accompanied by a change in the relative proportions of two chemical forms of the dye, each with a different fluorescence lifetime, which were found to co-exist in solution. From single-molecule fluorescence measurements, it was found that the fluorescence intensity and photobleaching time (on-time) of each CG-1 molecule was invariant with [Ca²⁺] and that changes in ensemble fluorescence intensity simply correlates with the number of fluorescent molecules in solution. These results are compared with that of the related system, Calcium Green 2 (CG-2), and the mechanisms of operation of these two indicator dyes are discussed.     

Photobleaching as a method of increasing the accuracy in measuring carotenoid concentration in human skin by Raman spectroscopy

It was shown experimentally that the effect of photobleaching in noninvasive measurement of the Raman spectra of light used in determining the carotenoid concentration in human skin can be used to increase measurement accuracy. Increased accuracy occurs as a consequence of a decrease in the measurable Raman spectra of the wideband fluorescent background intensity when a sample is irradiated by laser radiation. Furthermore, it was demonstrated that, for the spectra of skin from nine volunteers, the fluorescent background intensity can be decreased on average by a factor of 1.4, which leads to an increase in the signal-to-noise ratio for Raman lines of skin carotenoids by a factor of 1.2 on average. The kinetics of photobleaching of humans can be described by biexponential decay with a correlation coefficient close to unity, which agrees with the presented theoretical calculations.     

Scanning near-field optical microscopy-based study of local dynamics of receptor-ligand interactions at the single molecule level

A scanning near-field optical microscope (SNOM)—based modification of the method to study the dynamics of single molecule receptor—ligand interactions exploiting the fluorescence imaging by total internal reflection fluorescence microscopy is introduced. The main advantage of this approach consists in the possibility to study the single molecule interaction dynamics with a subwavelength spatial resolution and a submillisecond time resolution. Additionally, due to the much smaller irradiation area and some other technical features, such a modification enables to enlarge the scope of the receptor—ligand pairs to be investigated and to improve the temporal resolution. We briefly discuss corresponding experimental set up with a special accent on the SNOM operation in liquid and present some preliminary results of related investigations.     

稳态荧光探针法测定Tween系列非离子表面活性剂临界胶束浓度

以芘为荧光探针,测定了不同条件下的Tween系列表面活性剂增溶芘后的稳态荧光光谱,建立了Tween非离子表面活性剂的临界胶束浓度(CMC)的测试方法,研究了影响非离子表面活性剂临界胶束浓度的因素。根据I338/I333与Tween浓度的变化关系,可得到Tween20、40、60、80的临界胶束浓度分别为5.1×10-5、3.7×10-5、3.1×10-5、8×10-6mol/L。结果表明,同系列的非离子表面活性剂的分子结构对其临界胶束浓度有一定影响。同时,制备温度和外部添加的试剂如无机盐、乙醇、丙三醇均影响其临界胶束浓度。     

适配体传感法快速测定啤酒中赭曲霉毒素A

应用自组装方式,构建了金纳米粒子/核酸适体/氨基碳量子点荧光传感赭曲霉毒素A高灵敏检测方法。在pH 3.0酒石酸-HCl缓冲溶液中,巯基修饰的赭曲霉毒素A核酸适体在金纳米粒子表面自组装,形成金纳米粒子/核酸适体复合物,再在pH 7.0的磷酸盐缓冲溶液中,氨基碳量子点在金纳米粒子/核酸适体复合物上自组装,形成金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系。金纳米粒子的摩尔吸光系数大、能带宽使其具有强烈的荧光猝灭功能,氨基碳量子点形成金纳米粒子/核酸适体/氨基碳量子点后发生荧光猝灭, 此时体系的荧光为背景荧光,其强度记为F₀;由于金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系中核酸适体对赭曲霉毒素A具有特异性识别与结合功能,向金纳米粒子/核酸适体/氨基碳量子点复合物荧光传感检测体系溶液中加入赭曲霉毒素A后,赭曲霉毒素A则与复合物中核酸适体立即发生特异性结合并释放出氨基碳量子点,体系荧光恢复,其荧光强度记为F。依据体系荧光强度的变化(F-F₀)与赭曲霉毒素A浓度之间的关系,建立赭曲霉毒素A核酸适体荧光传感检测方法。研究了金纳米粒子和核酸适体摩尔比、孵化时间、pH等因素对传感器性能的影响,确定了最优条件为金纳米粒子∶核酸适体t为1∶190、孵化时间为6 min、pH 7.0时;在最优条件下,赭曲霉毒素A浓度在0.005～1.00 ng·mL-1范围与体系荧光强度变化呈良好线性关系,线性回归方程为：F-F₀=6.499+211.6c(c为赭曲霉毒素A的浓度,单位：ng·mL-1),相关系数r为0.995 5,按3倍标准差与工作曲线的斜率的比值(3σ/k)计算,得检测限为3 pg·mL-1。在实际样品中的回收率在93.3%～108.9%,相对标准偏差小于5%,能满足啤酒样品中赭曲霉毒素A快速检测要求。对13个市售啤酒样品进行检测,其中6个样品检出赭曲霉毒素A,污染率为46.15%。受污染样品的赭曲霉毒素A含量在0.008～0.63 ng·mL-1范围。该荧光传感法检测赭曲霉毒素A具有灵敏性好、特异性高、常见真菌毒素无干扰、方法简单、快速,便于大众化推广应用的优点。