Fluorescence photobleaching to evaluate flow velocity and hydrodynamic dispersion in nanoslits

Velocity measurement is a key issue when studying flows below the micron scale, due to the lack of sensitivity of conventional detection techniques. We present an approach based on fluorescence photobleaching to evaluate flow velocity at the nanoscale by direct visualization. Solutions containing a fluorescent dye are injected into nanoslits. A photobleached line, created through laser beam illumination, moves through the channel due to the fluid flow. The velocity and effective diffusion coefficient are calculated from the temporal data of the line position and width respectively. The measurable velocity range is only limited by the diffusion rate of the fluorescent dye for low velocities and by the apparition of Taylor dispersion for high velocities. By controlling the pressure drop and measuring the velocity, we determine the fluid viscosity. The photobleached line spreads in time due to molecular diffusion and Taylor hydrodynamic dispersion. By taking into account the finite spatial and temporal extensions of the bleaching under flow, we determine the effective diffusion coefficient, which we find to be in good agreement with the expression of the two dimensional Taylor-Aris dispersion coefficient. Finally we analyze and discuss the role of the finite width of the rectangular slit on hydrodynamic dispersion.     

Circumvention of fluorophore photobleaching in fluorescence fluctuation experiments: a beam scanning approach.

Photobleaching is a fluorophore-damaging process that commonly afflicts single-molecule fluorescence studies. It becomes an especially severe problem in fluorescence fluctuation experiments when studying slowly diffusing particles. One way to circumvent this problem is to use beam scanning to decrease the residence time of the fluorophores in the excitation volume. We report a systematic study of the effects of circular beam scanning on the photobleaching of fluorescent particles as observed in single-photon excitation fluorescence fluctuation experiments. We start by deriving a simple expression relating the average detected fluorescence to the photobleaching cross section of the fluorophores. We then perform numerical calculations of the spatial distribution of fluorescent particles in order to understand under which conditions beam scanning can prevent the formation of a photobleaching hole. To support these predictions, we show experimental results obtained for large unilamellar vesicles containing a small amount of the fluorescent lipophilic tracer DiD. We establish the required scanning radius and frequency range in order to obtain sufficient reduction of the photobleaching effect for that system. From the detected increase in fluorescence upon increase in scanning speed, we estimate the photobleaching cross section of DiD.     

Enhancing Fluorescence Brightness: Effect of Reverse Intersystem Crossing Studied by Fluorescence Fluctuation Spectroscopy

Experiments based on fluorescence detection are limited by the population of the fluorescence marker’s long‐lived dark triplet state, leading to pronounced photobleaching reactions and blinking which reduces the average fluorescence signal obtained per time interval. By irradiation with a second, red‐shifted laser line, we initiate reverse intersystem crossing (ReISC) which enhances the fluorescence signal of common fluorophores up to a factor of 14. The reverse intersystem crossing from the triplet state back to the singlet system is achieved by photoexcitation to higher‐excited triplet states, which are, however, prone to photobleaching. We gain insights into the competing pathways of ReISC and photobleaching. The relative efficiencies of these two pathways and the triplet lifetime determine the achievable fluorescence enhancement, which varies strongly with the choice of dye, excitation irradiance and wavelength, and with environmental conditions. The study of ReISC not only results in a better understanding of a fluorescent label’s photophysics, but the method is a possible approach to optimize fluorescence emission in experiments, where signal strength is a critical parameter.     

Photobleaching of reduced nicotinamide adenine dinucleotide and the development of highly fluorescent lesions in rat basophilic leukemia cells during multiphoton microscopy

Endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence provides an intrinsic indicator of the cellular metabolic state, but prolonged monitoring is limited by photobleaching and/or phototoxicity. Multiphoton excitation of NADH by ultrashort, 740-nm laser pulses provides a significant improvement over UV excitation by eliminating peripheral photobleaching; however, molecules within the subfemtoliter excitation volume remain susceptible. We have investigated the photophysical mechanisms responsible for multiphoton photobleaching of NADH in living cells to permit the imaging technique to be optimized. The loss of fluorescence because of multiphoton photobleaching was measured by repetitively imaging individual planes within rat basophilic leukemia cells. The photobleaching rate was proportional to the fourth power of the laser intensity. Based on these measurements, we propose a double-biphotonic, four-photon photobleaching mechanism and estimate the quantum yield of photobleaching of intracellular NADH to be 0.0073 +/- 0.0002 by this mechanism. In addition to photobleaching, the development of bright, punctate fluorescent lesions can also be observed. The frequency of lesion formation also increased approximately as the fourth power of the laser intensity after an intensity-dependent threshold number of images had been exceeded. The consequences for two-photon metabolic imaging are discussed.     

Many-photon dynamics of photobleaching

A detailed dynamical theory of photobleaching by periodical sequences of laser pulses is presented. The theory is used for interpretation of recent experiments with pyrylium salts. Our simulations are based on first-principles simulations of photoabsorption cross-sections and on empirical rate constants. Two competitive channels of photobleaching, namely, photobleaching from the lowest excited singlet and triplet states and from higher excited states, are found to explain different intensity dependences of the photobleaching rates in different samples. The process includes two-photon excitation from the ground state to the first or second excited singlet states and one-photon excitation from the first singlet or triplet states to higher excited states. The fluorescence follows double-exponential dynamics with two characteristic times. The first and the shorter one is the equilibrium settling time between the ground and the lowest triplet states. The second characteristic time, the time of photobleaching, is responsible for the long-term dynamics. The effective rate of photobleaching from the first excited singlet and lowest triplet states depends differently on the irradiance in comparison with the photobleaching in higher states. The first channel is characterized by a quadratic intensity dependence in contrast to the second channel that shows a cubic dependence. The competition between these photobleaching channels is very sensitive to the rate constants as well as to the repetition rate, the pulse duration, and the peak intensity. The double-exponential decay of the fluorescence is explained by the spatial inhomogeneity of the light beam. The findings in this work are discussed in terms of the possibility of using many-photon-induced photobleaching for new three-dimensional read-write devices.     

Nonexponential statistics of fluorescence photobleaching

In this paper, I consider theoretical models of the decay via photobleaching of a sample of surface-immobilized fluorescent molecules excited by a spatially varying laser intensity profile. I show that, with mild restrictions on the photobleaching mechanism, the fluorescence decay measured in a nonuniform excitation profile is always nonexponential. Under the same conditions, the fluorescence decay can always be approximated by a discrete sum of exponentials. A particular example is given in which a homogeneous population of fluorophores with a single (intensity-dependent) photobleaching lifetime, when illuminated by a Gaussian laser, exhibits power law fluorescence decay at long times. These results indicate that the observation of multiple exponentials in single molecule or ensemble photobleaching lifetime measurements can arise solely as an artifact of a spatially varying laser profile and is not necessarily indicative of heterogeneity in molecular internal states, conformation, or local environment.     

Dye aggregation and influence of pre-micelles on heterogeneous catalysis: A photophysical approach

Common cationic dyes used for laser and fluorescent probes present low solubility in water. In order to increase the dye concentration in aqueous solutions, anionic surfactant can be added. The strong interaction between anionic surfactant and cationic dye can affect drastically the dye absorption and fluorescence properties. Here we observed that the fluorescence of the species in aqueous solution is maximized at condition of complete micellization of surfactants at critical micelle concentration (CMC). In addition, combined measurements of absorption, emission and fluorescence lifetime provide fundamental information on the critical concentration of H-aggregates formation and monomer separation, induced by pre-micelles and homomicelles on different surfactant sodium dodecylsulphate (SDS) concentration. The experimental results show how to find precisely the critical concentration of H-aggregates by optical method in two different xanthene-derived molecules: rhodamine 6G and rhodamine B. The adequate transference of electron from excited dye to the conduction band of semiconductor (TiO₂) promotes the creation of reactive species that provides the degradation of dye with advantage of use of irradiation in the visible region and strong photobleaching with direct exposure to the visible light irradiation in a scale of time of 10 min.     

SPONTANEOUS RECOVERY OF FLUORESCENCE BY PHOTOBLEACHED SURFACE-ADSORBED PROTEINS

Abstract-Fluorescence photobleaching of a carboxyfiuorescein-labeled protein (erythrocyte cytoskel-etal protein 4.1) immobilized on bare glass is found to be spontaneously reversible, provided that the sample is deoxygenated. After a short (hundredths of seconds) photobleaching laser flash, the subsequent fluorescence excited by a dim probe beam partly recovers on a long (tenths of second) time scale, even in the absence of chemical exchange or diffusion processes. Neither the fraction of the fluorescence that bleaches reversibly nor its recovery rate is a strong function of fluorophore surface concentration. At a fixed surface concentration, the reversibly photobleached fraction and its recovery rate decreases with increasing duration or intensity of the bleaching flash. On the other hand, nondeoxygenated air-equilibrated samples exhibit almost total irreversible bleaching on this time scale. Quantitative fluorescence microscopy experiments occasionally require deoxygenation to avoid photochemical crosslinking or photobleaching or to enhance the triplet state population. The observations presented here indicate that fluorescence recovery after photobleaching (FRAP) experiments performed under deoxygenated conditions for measuring diffusion or chemical kinetics should be interpreted with caution: fluorescence recoveries may be due to intrinsic photochemical processes rather than fluorophore mobility. The recovery effect appears too slow to be ascribed simply to a relaxation of a triplet state; other possible explanations are offered.     

Competition between photobleaching and fluorescence increase of photosensitizing porphyrins and tetrasulphonated chloroaluminiumphthalocyanine

The time-dependent fluorescence changes of photosensitizing porphyrins and tetrasulphonated chloroaluminiumphthalocyanine (A1C1SPc) were measured at different intracellular sites using video-enhanced microscopy and image processing. To obtain variations in intracellular fluorescence intensity, different radiant exposures of a Kr+ laser-pumped dye laser were delivered via a 600 microns plastic-clad silica fibre connected to the microscope. During irradiation, competition between photobleaching and fluorescence increase of the different dyes was observed. The porphyrins normally showed photobleaching, which was dependent on the sensitizer and its specific accumulation within the cell. Photobleaching was less pronounced for hydrophilic uroporphyrin than for more hydrophobic dyes. In contrast with an almost exponential decrease in porphyrin fluorescence with increasing light dose, the fluorescence intensity of A1C1SPc significantly increased at the beginning of irradiation, and could be correlated with intracellular deaggregation.     

Picosecond multidimensional fluorescence spectroscopy: a tool to measure real-time protein dynamics during function

Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-resolved fluorescence in combination with site-directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico- to nanoseconds time scale. Time-resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is light-activated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPC-module. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket.     

Photobleaching of Arterial Fluorescent Compounds: Characterization of Elastin, Collagen and Cholesterol Time-resolved Spectra during Prolonged Ultraviolet Irradiation

To study the photobleaching of the main fluorescent compounds of the arterial wall, we repeatedly measured the time-resolved fluorescence of elastin, collagen and cholesterol during 560 s of excitation with nitrogen laser pulses. Three fluence rate levels were used: 0.72, 7.25 and 21.75 microW/mm2. The irradiation-related changes of the fluorescence intensity and of the time-resolved fluorescence decay constants were characterized for the emission at 390, 430 and 470 nm. The fluorescence intensity at 390 nm decreased by 25-35% when the fluence delivered was 4 mJ/mm2, a common value in fluorescence studies of the arterial wall. Cholesterol fluorescence photobleached the most, and elastin fluorescence photobleached the least. Photobleaching was most intense at 390 nm and least intense at 470 nm such that the emission spectra of the three compounds were markedly distorted by photobleaching. The time-resolved decay constants and the fluorescence lifetime were not altered by irradiation when the fluence was below 4 mJ/mm2. The spectral distortions associated with photobleaching complicate the interpretation of arterial wall fluorescence in terms of tissue content in elastin, collagen and cholesterol. Use of the time-dependent features of the emission that are not altered by photobleaching should increase the accuracy of arterial wall analysis by fluorescence spectroscopy.     

Photobleaching of asymmetric cyanines used for fluorescence imaging of single DNA molecules

The photobleaching of the cyanine dyes YO and YOYO has been investigated for both free and DNA-bound dyes, using absorption and fluorescence spectroscopy coupled with fluorescence microscopy. For the free dyes, the nature of the reactive species involved in the photodegradation process is different for the monomer and the dimer, as shown by scavenger studies. For DNA-bound dyes, photoinduced fading of the visible absorption band occurs by different pathways depending on the drug binding mode and can be attenuated by appropriate scavengers. However, none of these scavengers were found to have any significant effect on the photobleaching of dye fluorescence. It appears that the reduction of fluorescence intensity comes from a quenching of the dye fluorescence by modified DNA bases, possibly 8-oxo-7,8-dihydro-2'-deoxyguanosine.     

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

The development of stimulated emission depletion (STED) microscopy represented a major breakthrough in cellular and molecular biology. However, the intense laser beams required for both excitation and STED usually provoke rapid photobleaching of fluorescent molecular probes, which significantly limits the performance and practical utility of STED microscopy. We herein developed a photoresistant fluorescent dye C‐Naphox as a practical tool for STED imaging. With excitation using either a λ=405 or 488 nm laser in protic solvents, C‐Naphox exhibited an intense red/orange fluorescence (quantum yield Φ_F>0.7) with a large Stokes shift (circa 5900 cm^?1). Even after irradiation with a Xe lamp (300 W, λ_ex=460 nm, full width at half maximum (FWHM)=11 nm) for 12 hours, 99.5 % of C‐Naphox remained intact. The high photoresistance of C‐Naphox allowed repeated STED imaging of HeLa cells. Even after recording 50 STED images, 83 % of the initial fluorescence intensity persisted.     

Fluorescence correlation spectroscopy for flow rate imaging and monitoring--optimization, limitations and artifacts

We recently demonstrated a new method for mapping fluid velocities in 3 dimensions and with exceptionally high spatial resolution for the characterization of flow in microfluidic devices. In the method, a colloidal suspension containing fluorescent tracer particles, dye doped polymer spheres, is pumped through a microchannel and confocal microscopy combined with fluorescence correlation spectroscopy is used to measure fluid velocities. In this report, we further characterize the technique and report on optimizations that allow a 5-fold increase in speed of single point velocity measurements. This increase in measurement speed will yield a 25 fold reduction in the time needed to collect a complete velocity image. The precision of measured velocities was characterized as a function of tracer particle concentration, measurement time, and fluid velocity. In addition, we confirm the linearity of the measurement method (velocity vs. applied pressure) over a range of velocities spanning four orders of magnitude. Furthermore, we demonstrate that an artifact in velocity measurements using fluorescence correlation spectroscopy (FCS) that was interpreted by others as being caused by optical trapping forces is actually an artifact caused by detector saturation and can be avoided by careful choice of experimental conditions.     

筛选抗癌药物的荧光实验法

筛选抗癌药物常用的体外肿瘤系统方法有甲烯蓝法、染色法、细胞呼吸法等,这些方法需要取肿瘤细胞进行实验,时间较长。顺铂是很好的抗癌药物,Sohell等用DNA-溴化乙啶(EthBr)荧光体系研究了铂络合物与DNA的作用,认为该体系可以半定量地说明铂络合物的抗癌性。本文研究发现一些络合物-DNA-EthBr体系的荧光大小与络合物的抗癌性有关,可用荧光法筛选抗癌药物。其可靠性远比甲烯蓝法高,方法简单、快速,且假阳性少。     

Synthesis of fluorescent silica-coated gibbsite platelets

We describe the fluorescent labeling of gibbsite particles. Gibbsite particles are first stabilized with polyvinyl pyrrolidone. Subsequently the particles are covered with a silica layer in which a fluorescent dye is incorporated. Both fluorescein and rhodamine dyes have been used. The fluorescent labeling is applicable to gibbsite particles of various sizes. Particles are transferred to dimethyl formamide by vacuum distillation after dialysis. These particles are used for confocal scanning laser microscopy and confocal fluorescence-recovery after photobleaching.     

Distribution of single molecules in polymer thin films

Distribution of fluorescent dye molecules in polymer thin (100 nm) films was investigated using far-field single-molecule video microscopy, by varying concentrations of dye molecules mixed in the polymer. Histograms of fluorescence photocounts of individual fluorescent spots showed wide distribution, varying in the number of fluorescent spots composed of one, two, three or group of molecules. The number of the molecules present in the fluorescent spots was also ascertained by fluorescence photobleaching experiments. Photocounts associated with maxima of the histograms were found to be independent of the concentrations; however, the number of occurrences associated with more than one molecule decreased with decreasing concentration. By reducing concentration as well as by mixing dye molecules into a polymer solution, fluorescent spots grouping more than one molecule were separated considerably into fluorescent spots including a single-molecule.     

Laser-induced fluorescence of trapped gas-phase molecular ions generated by internal source matrix-assisted laser desorption/ionization in a Fourier transform ion cyclotron resonance mass spectrometer

The combination of laser-induced fluorescence with mass spectrometry opens up new possibilities both for detection purposes and for structural studies of trapped biomolecular ions in the gas phase. However, this approach is experimentally very challenging, and only a handful of studies have been reported so far. In this contribution, a novel scheme for laser-induced fluorescence measurements of ions trapped inside a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer will be introduced. It is based on an open FT-ICR cell design, continuous wave axial excitation of the fluorescence, orthogonal photon collection by fiber optics, and single photon counting detection. Rhodamine 6G ions generated by an internal matrix-assisted laser desorption/ionization source were used to develop and test the set-up. Due to photobleaching processes, the excitation laser power and the observation time window have to be carefully optimized. An ion tomography method was used to align the excitation laser. Potential applications for studying the gas-phase structure of fluorescent biomolecular ions and for investigating fluorescence resonance energy transfer of donor-acceptor pairs will be presented.