Heterogeneous photobleaching in confocal microscopy

Photobleaching was studied during recording of confocal scanning laser microscopy. Studies on fluorescent gels of FITC-labeled dextran were used to evaluate differential bleaching along thez-axis. Differential bleaching along the z-axis was observed and it was seen that this was related to the numerical aperture of the objective in use. This points to the conclusion that photon energy flux density is an important parameter in photobleaching. To check if photon energy flux density heterogeneity is affected by local variation in the refractive index of the sample, photobleaching rates were calculated for different fluorescent objects (sections of seeds, animal cells stained with nuclear stains, immunocytochemistry preparations) and a pronounced similarity was found between photobleaching rates and DIC images.     

High Probe Intensity Photobleaching Measurement of Lateral Diffusion in Cell Membranes

Lateral diffusion measurements, most commonly accomplished through Fluorescence Photobleaching Recovery (FPR or FRAP), provide important information on cell membrane molecules' size, environment and participation in intermolecular interactions. However, serious difficulties arise when these techniques are applied to weakly expressed proteins of either of two types: fusions of membrane receptors with visible fluorescent proteins or membrane molecules on autofluorescent cells. To achieve adequate sensitivity in these cases, techniques such as interference fringe FPR are needed. However, in such measurements, cytoplasmic species contribute to the fluorescence recovery signal and thus yield diffusion parameters not properly representing the small number of surface molecules. A new method helps eliminate these difficulties. High Probe Intensity (HPI)-FPR measurements retain the intrinsic confocality of spot measurements to eliminate interference from fluorescent cytoplasmic species. However, HPI-FPR methods lift the previous requirement that FPR procedures be performed at probe beam intensities low enough to not induce bleaching in samples during measurements. The high probe intensities now employed provide much larger fluorescence signals and thus more information on molecular diffusion from each measurement. We report successful measurement of membrane dynamics by this technique.     

An Alternate Solution of Fluorescence Recovery Kinetics after Spot-Bleaching for Measuring Diffusion Coefficients. 2. Diffusion of Fluorescein in Aqueous Sucrose Solutions

The traditional analysis of the fluorescence recovery kinetics after spot bleaching yields expressions for the diffusion coefficient of the probe that are not suitable for linear fittings. In a previous work we developed an improved recovery function that is a better alternative for data analysis. To illustrate its application to real cases and compare it with the previous data treatment, we measured the time response of fluorescein in aqueous sucrose solutions, covering the unsaturated and the supercooled region, where decoupling between diffusion and viscosity is observed. The results are compared with the mobility of different types of solutes in aqueous sucrose solutions and are discussed in terms of the classical hydrodynamic model.     

光漂白全过程中聚合物薄膜折射率和厚度的实时分析

在基于衰减全反射原理的基础上提出一种新的测量方法, 动态研究漂白动力学过程, 即利用波导的衰减全反射吸收峰对聚合物材料的折射率和厚度敏感的特性, 实时测量聚合物材料的折射率和厚度. 实验系统采用CCD摄像头将标志波导模式的一组暗线显示在计算机屏幕上, 然后根据暗线的移动, 可以精确测量每一时刻波导薄膜的折射率和膜厚. 利用这种技术, 对聚合物薄膜的光漂白过程进行了实时监控. 发现在光漂白全过程中, 聚合物薄膜的折射率和厚度的变化同时存在化学和物理两种变化过程.     

Poly-3BCMU 薄膜光漂白过程的理论模型

本文报道了使用两种不同漂白光源时,Poly-3BCMU薄膜的光漂白过程,并建立了描述漂白过程的理论模型.最后讨论了采用光漂白方法制备导波微光学元件的可能性.     

VISIBLE LIGHT PHOTOPOLYMERIZATION OF METHYL METHACRYLATE COINITIATED WITH TITANOCENE AND KETOCOUMARIN DYE*

The photosensitive system which can initiate methyl methacrylate with visible light was composedof compound 1 bis(η-5-cyclopentadienyl)-bis[2,6-difluoro-3-(1-H-pyrrolyl)phenyl]titanium (titanocene) andcompound 2 [(3,3'-carbonylbis(7-diethylamino coumarin)] (ketocoumarin dye). The high photosensitiveinitiating efficiency of this photosensitive system could be very promising for efficient system for laser (Ar~+488 nm) to plate and photocuring for thick coating and ink. The variation of UV-visible spectrum ofcompound 2 during irradiation indicates that photolysis of compound 2 is through its triplet state and it canbe quenched by O_2. The much quicker photobleaching of the photosensitive system suggests that there existscertain quick electron transfer reaction between compounds 1 and 2.     

Photoinitiation rate profiles during polymerization of a dimethacrylate-based resin photoinitiated with camphorquinone/amine. Influence of initiator photobleaching rate

The photodecomposition of camphorquinone (CQ)/amine during the photo polymerization of a dimethacrylate-based resin under continuous irradiation was investigated in thick samples. The global CQ photoconsumption was measured by monitoring the decrease in light absorption as a function of irradiation time and the kinetics were satisfactory fitted to a first order expression where the rate constant of photobleaching was proportional to the irradiation intensity. In a thick sample, the photobleaching of the photoinitiator is accompanied by a deeper penetration of the light through the underlying layers. These gradients of photoinitiator concentration, light intensity and photoinitiation rate along the path of irradiation were calculated. The photodecomposition reaction was spatially inhomogeneous and the degree of nonuniformity increased with increased initial sample absorbance. The influence of the photobleaching process on the polymerization reaction was examined. The photobleaching rate of CQ was much slower than the polymerization rate and only 20% of the initial amount of CQ was consumed before the polymerization reaction had almost ceased. Results obtained in this research highlight the inherent interlinking of light attenuation and photobleaching rate in bulk polymerizing systems.     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

聚合物PMMA/DR1薄膜棱镜的制备

本文研究了用光漂白方法制备ＰＭＭＡ／ＤＲ１聚合物光棱镜的方法，测量了这种波导棱镜对１．０６４μｍ光的ＴＥ模和ＴＭ模在波导内传播光束的方向改变，并从理论上对测量结果进行了分析。     

Spectrophotometric and spectrofluorimetric studies of novel heterocyclic Schiff base dyes

Novel heterocyclic Schiff base dyes were prepared by the reaction of salicylaldehyde/2-Hydroxy-1-naphthaldehyde with different heterocyclic amines. Results of the newly synthesized compounds established by FT-IR, ¹H-NMR, ¹³C-NMR and GC–MS spectroscopic experiments were consistent with their chemical structures. Resulted heterocyclic Schiff base dyes were found to be pure from data obtained by the elemental analysis. In addition, spectrophotometric and spectrofluorimetric studies showed that these dyes were good absorbent and fluorescent. Fluorescence polarity study data revealed that some of these compounds were sensitive to the polarity of the microenvironment provided by different solvents. Time-based fluorescence steady-state measurements also showed that these heterocyclic Schiff base dyes have high photostability against photobleaching.