Fluorescence microspectroscopy technique for the study of intracellular protoporphyrin IX dynamics

We describe a technique designed to monitor the fluorescence dynamics of photosensitizers used in photodynamic therapy (PDT) at micrometer-scale locations within individual formalin-fixed cells. The accumulation of protoporphyrin IX (PpIX) within keratinocytes and fibroblasts. following incubation with 5-aminolaevulinic acid (ALA), is shown to be dependent upon both incubation time and cell proliferation status. Also, the process of photobleaching within these cells is demonstrated via the depletion in PpIX fluorescence emission during exposure to 532 nm light. All spectra show a progressive reduction of the 634 nm PpIX peak, following a bi-exponential decay that is consistent with a singlet oxygen mediated process. The rate of photobleaching, when plotted as a function of light dose, increases with reduced incident laser power. The generation of the hydroxyaldehyde-chlorin photoproduct (photoprotoporphyrin), as monitored by the increase in fluorescence emission centered on 672 nm, is also greatest when the lowest laser power is applied. When light is delivered in two fractions, PpIX fluorescence recovers during the dark period and there is an increase in bleaching rate at the onset of the second exposure. These results are qualitatively consistent with measurements performed in vivo, which demonstrate that the photodynamic dose is dependent upon fluence rate and oxygen status.     

Energy transfer and photodegradation of a Perylene Orange:LDS821 system in poly(methyl methacrylate)

The luminescence of Perylene Orange and LDS821 in poly(methyl methacrylate) (PMMA) following a 532 nm irradiation yielded information on photodegradation and energy transfer. The irradiation of the Perylene Orange/LDS821/PMMA films resulted in (i) a maximum in the Perylene Orange fluorescence photodegradation profile and (ii) an enhancement of the LDS821 fluorescence relative to the LDS821/PMMA films. These results are attributed to an energy transfer from the Perylene Orange to the LDS821 with an energy transfer rate constant of 5.1+/- 0.75 (2sigma) x 10(11) M(-1) s(-1) and a F?rster critical radius of 65.7 A. Fluorescence half-quenching and time-resolved fluorescence measurements attributed energy transfer to the F?rster energy transfer with minor contributions of radiative energy transfer.     

On photo-oxidation of single molecules

Fluorescence and fluorescence excitation spectra of terrylene in 9,10-dibromoanthracene matrix were studied at 5 K. Characteristic singlet oxygen phosphorescence, O₂(¹Δ_g) → O₂(³Σ_g) with the line maximum at 1272.5 nm, was observed after excitation of terrylene molecules embedded into one of its two sites. It indicates that singlet oxygen, precursor in photo-oxidation of aromatic molecules, can be created only for those oxygen molecules which can form weak van der Waals complex with terrylene. According to quantum-chemical calculations terrylene and oxygen can form complex with the bonding energy 141 cm⁻¹, in which oxygen molecule is located along the long axis of terrylene, ∼0.3 nm apart, with the molecular axis inclined by 110°.     

Singlet oxygen and dye-triplet-state quenching in solid-state dye lasers consisting of pyrromethene 567-doped poly(methyl methacrylate)

Solid-state dye lasers based on poly(methyl methacrylate) (PMMA) doped with Pyrromethene 567 dye (P567) have been investigated. The preparation techniques employed provided high photostability and laser damage threshold for P567 in pure PMMA with 270,000 pulses emitted before the conversion efficiency fell to half its initial value for a pump fluence of 0.16 J cm(-2). When PMMA was modified with 1, 4-diazobicyclo [2, 2, 2] octane singlet oxygen quencher, the longevity increased to 550,000 pulses, corresponding to a normalized photostability of 270 GJ mol(-1). Modification of PMMA with a triplet quencher (perylene) yielded no improvement, but in ethanol solutions both additives enhanced photostability. It is possible that in PMMA, stabilization by means of triplet quenching that depends on dye diffusion is prevented but that stabilization by means of singlet oxygen quenching that depends on the faster oxygen diffusion rate will succeed.     

A Novel Theranostic Nanoprobe for In Vivo Singlet Oxygen Detection and Real‐Time Dose–Effect Relationship Monitoring in Photodynamic Therapy

Singlet oxygen, as the main member of reactive oxygen species, plays a significant role in cancer photodynamic therapy. However, the in vivo real‐time detection of singlet oxygen remains challenging. In this work, a Förster resonance energy transfer (FRET)‐based upconversion nanoplatform for monitoring the singlet oxygen in living systems is developed, with the ability to evaluate the in vivo dose–effect relationship between singlet oxygen and photodynamic therapy (PDT) efficacy. In details, this nanoplatform is composed of core–shell upconversion nanoparticles (UCNPs), photosensitizer MC540, NIR dye IR‐820, and poly(acryl amine) PAA‐octylamine, where the UCNPs serve as an energy donor while IR‐820 serves as an energy acceptor. The nanoparticles are found to sensitively reflect the singlet oxygen levels generated in the tumor tissues during PDT, by luminescence intensity changes of UNCPs at 800 nm emission. Furthermore, it could also enable tumor treatment with satisfactory biocompatibility. To the best knowledge, this is the first report of a theranostic nanoplatform with the ability to formulate the in vivo dose–effect relationship between singlet oxygen and PDT efficacy and to achieve tumor treatment at the same time. This work might also provide an executable strategy to evaluate photodynamic therapeutic efficacy based on singlet oxygen pathway.     

UV-light induced photodegradation of 17alpha-ethynylestradiol in aqueous solutions

The photodegradation of 17alpha-ethynylestradiol (EE2) in aqueous solutions induced by UV-light was preliminarily studied in this paper by means of fluorescence, UV and infrared spectra. The result suggested that EE2 in aqueous solutions underwent photodegradation under irradiation with UV disinfection lamp (lambda = 254 nm, 30 W), but the photodegradation was not observed under high pressure mercury lamp (lambda > or = 365 nm, 250 W). The photodegradation of 1.6-20.0 mg/l EE2 in aqueous solutions at a given initial pH value of 6.8 was pseudo-first order reaction. Increasing the initial concentration of EE2 lowered the photodegradation rate. The photodegradation rate of EE2 reached the lowest value at pH about 5.0, higher pH values of 6.0-8.0 benefited the photodegradation. Ferric ions can promote the photodegradation of EE2 in aqueous solutions at pH value of 2.0-5.0.     

8-aminoquinoline functionalized silica nanoparticles: a fluorescent nanosensor for detection of divalent zinc in aqueous and in yeast cell suspension

Zinc is one of the most important transition metal of physiological importance, existing primarily as a divalent cation. A number of sensors have been developed for Zn(II) detection. Here, we present a novel fluorescent nanosensor for Zn(II) detection using a derivative of 8-aminoquinoline (N-(quinolin-8-yl)-2-(3 (triethoxysilyl)propylamino)acetamide (QTEPA) grafted on silica nanoparticles (SiNPs). These functionalized SiNPs were used to demonstrate specific detection of Zn(II) in tris-HCl buffer (pH 7.22), in yeast cell (Saccharomyces cerevisiae) suspension, and in tap water. The silane QTEPA, SiNPs and final product were characterized using solution and solid state nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption spectroscopy, transmission electron microscopy, elemental analysis, thermogravimetric techniques, and fluorescence spectroscopy. The nanosensor shows almost 2.8-fold fluorescence emission enhancement and about 55 nm red-shift upon excitation with 330 ± 5 nm wavelength in presence of 1 μM Zn(II) ions in tris-HCl (pH 7.22). The presence of other metal ions has no observable effect on the sensitivity and selectivity of nanosensor. This sensor selectively detects Zn(II) ions with submicromolar detection to a limit of 0.1 μM. The sensor shows good applicability in the determination of Zn(II) in tris-HCl buffer and yeast cell environment. Further, it shows enhancement in fluorescence intensity in tap water samples.     

苝酰亚胺类化合物的功能改性及光谱性能研究

本文合成了三种含有羧基的苝酰亚胺化合物，并研究了其紫外-可见吸收光谱及荧光光谱的特征；研究表明，苝环上1，6，7，12位取代基的引入，使得所合成的苝酰亚胺类化合物的最大吸收波长分别红移了50nm、49nm；Stokes位移变大30、31nm。而荧光量子产率的下降，但不是很明显。     

Remote fluorescence imaging of dynamic concentration profiles with micrometer resolution using a coherent optical fiber bundle

Dynamic concentration profiles within the diffusion layer of an electrode were imaged in situ using fluorescence detection through a multichannel imaging fiber. In this work, a coherent optical fiber bundle is positioned orthogonal to the surface of an electrode and is used to report spatial and temporal micrometric changes in the fluorescence intensity of an initial fluorescent species. The fluorescence signal is directly related to the local concentration of a redox fluorescent reagent, which is electrochemically modulated by the electrode. Fluorescence images are collected through the optical fiber bundle during the oxidation of tris(2,2'-bipyridine)ruthenium(II) to ruthenium(III) at a diffusion-limited rate and allow the concentration profiles of Ru(II) reagent to be monitored in situ as a function of time. Tris(2,2'-bipyridine)ruthenium(II) is excited at 485 nm and emits fluorescence at 605 nm, whereas the Ru(III) oxidation state is not fluorescent. Our experiments emphasize the influence of two parameters on the micrometer spatial resolution: the numerical aperture of optical fibers within the bundle and the Ru(II) bulk concentration. The extent of the volume probed by each individual fiber of the bundle is discussed qualitatively in terms of a primary inner-filter effect and refractive index gradient. Experimentally measured fluorescence intensity profiles were found to be in very good agreement with concentration profiles predicted upon considering planar diffusion and thus validate the concept of this new application of imaging fibers. The originality of this remote approach is to provide a global view of the entire diffusion layer at a given time through one single image and to allow the time expansion of the diffusion layer to be followed quantitatively in real time.     

Nanoporous p-terphenyl-polystyrene films containing perylene; fabrication,characterization and remarkable fluorescence resonance energy transfer based blue emitting properties

Strongly blue luminescent porous p-terphenyl-polystyrene films containing perylene was obtained on glass substrates using a simple Breath Figure method. The size and shape of porous with perylene load from scanning electron microscopy was seen to be decreased from 600 to 70 nm. However, the contact angle of films loading with perylene found to be increased with the decrease in pores size. This result is helped to propose a mechanism of Breath Figure formation. The fluorescence spectra of doped polystyrene (PS) films showed the characteristic monomer emission of perylene at 478 nm with simultaneous quenching of p-terphenyl fluorescence due to excitation energy transfer from p-terphenyl to perylene. The estimated values of the efficiency of energy transfer are found to be pore size dependant. The spectral results of quenching of p-terphenyl and simultaneously sensitization of perylene emission are in good agreements with the color seen in fluorescence microscope images and fluorescence life time measurements. The sensitization of blue emission from Breath Figure pattern of PS films is of current demand into the fabrication of micropatterning devices.     

First Demonstration of Gold Nanorods‐Mediated Photodynamic Therapeutic Destruction of Tumors via Near Infra‐Red Light Activation

Previously, a large volume of papers reports that gold nanorods (Au NRs) are able to effectively kill cancer cells upon high laser doses (usually 808 nm, 1–48 W/cm²) irradiation, leading to hyperthermia‐induced destruction of cancer cells, i.e, photothermal therapy (PTT) effects. Combination of Au NRs‐mediated PTT and organic photosensitizers‐mediated photodynamic therapy (PDT) were also reported to achieve synergistic PTT and PDT effects on killing cancer cells. Herein, we demonstrate for the first time that Au NRs alone can sensitize formation of singlet oxygen (¹O₂) and exert dramatic PDT effects on complete destrcution of tumors in mice under very low LED/laser doses of single photon NIR (915 nm, <130 mW/cm²) light excitation. By changing the NIR light excitation wavelengths, Au NRs‐mediated phototherapeutic effects can be switched from PDT to PTT or combination of both. Both PDT and PTT effects were confirmed by measurements of reactive oxygen species (ROS) and heat shock protein (HSP 70), singlet oxygen sensor green (SOSG) sensing, and sodium azide quenching in cellular experiments. In vivo mice experiments further show that the PDT effect via irradiation of Au NRs by 915 nm can destruct the B16F0 melanoma tumor in mice far more effectively than doxorubicin (a clinically used anti‐cancer drug) as well as the PTT effect (via irradiation of Au NRs by 780 nm light). In addition, we show that Au NRs can emit single photon‐induced fluorescence to illustrate their in vivo locations/distribution.     

Photosensitized degradation in water of the phenolic pesticides bromoxynil and dichlorophen in the presence of riboflavin, as a model of their natural photodecomposition in the environment

Within the context of environmentally friendly methods for the elimination of surface-water pollutants, the photodegradation of the phenolic pesticides bromoxynil (BXN) and dichlorophen (DCP) under simulated natural conditions has been studied. The work was done in the presence of the visible-light absorber photosensitizer riboflavin (Rf), usually present in trace quantities in natural waters. Under aerobic conditions, an efficient photooxidation of both pesticides was observed. The relatively intricate photochemical mechanism involves pesticide and oxygen consumption and, to a lesser extent, Rf degradation. The kinetic and mechanistic study supports that both H(2)O(2) and singlet molecular oxygen, O(2)((1)Δ(g)), are involved in the process. Kinetic data for the O(2)((1)Δ(g))-mediated oxidation indicate that BXN and DCP are photodegraded with this species faster than the parent compound phenol, very frequently employed as a model for aquatic contaminants, likely due to their lower pK(a) values. This observation allows the design of phenolic pesticides with different photodegradation rates under environmental conditions.     

Effect of plasticization on the photodegradation of poly (para-methoxystyrene) films

The influence of dibutyl phthalate, dioctyl phthalate, dimethyl terephthalate, diethyl terephthalate, and dioctyl terephthalate-plasticizers on poly (para-methoxy styrene) photodegradation was investigated by UV–vis, fluorescence, and FT-IR spectroscopic methods. The increase in irradiation time caused an increase in the absorption band of the polymer and an increase in the absorption of new band at longer wavelength, thus indicating a possibility of photodegradation of polymer films. The study on poly (para-methoxy styrene) photodegradation was also compared to varying phthalate and terephthalate plasticizers in order to provide an understanding of its photodegradation vis the presence of phthalates and terephthalates plasticizers. The decrease in the excimer fluorescence upon the increase in the time of exposure to UV-radiation provides an evidence for the degradation of polymeric chains through a chin scission processes. The increase in the photodegradation of the polymer increased with the increase in the bulkiness of added plasticizer molecule. IR-spectra of irradiated pure and blended polymer films indicated that the photodegradation of the polymer occurred, by the formation of new absorption bands, and the increase in the intensity of other bands.     

Photostability enhancement of Pyrromethene 567 and Perylene Orange in oxygen-free liquid and solid dye lasers

We investigated the effect of oxygen on the photostability of the laser dyes Pyrromethene 567, Perylene Orange, and Rhodamine 590 by determining their longevity of laser operation when pumped by the second harmonic of aQ -switched Nd:YAG laser. In solution, dissolved oxygen accelerated the photodegradation of Pyrromethene 567 and Perylene Orange but not Rhodamine 590. The photostability of Pyrromethene 567 was also found to be dependent on the solvent and on the lifetime of singlet oxygen. Deoxygenated Pyrromethene 567 doped polycom glass and modified poly(methyl methacrylate) (MPMMA) samples were tested for longevity of laser operation. A factor of 6 improvement in photostability was found for Pyrromethene 567 in MPMMA upon deoxygenation, and the total absorbed energy per mole of dye molecules to one-half output pulse energy was 36 GJ mol-1 .     

Modeling of Photochemical Reactions in a Focused Laser Beam,II

A method is described for obtaining the rate constant of the photodegradation process of fluorophores illuminated by a focused laser beam. The explicit kinetic equations, describing the population dynamics of excited singlet and triplet states, are averaged over the illuminated volume to describe the resulting fluorescence signal. The illumination is modulated at frequencies from 1 Hz to 100 Hz. Synchronous detection of the resulting fluorescence yields in-phase and quadrature components. The measurement of the ratio of quadrature to in-phase components at several power levels yields information on the photodegradation rate. Specifically it is shown that the data can be interpreted in a manner which yields the value of the photodegradation rate independently of other parameters entering the model. Experiments are performed with erythrosine B which has a large intersystem crossing rate to the triplet state. Measurements in solutions with different viscosities show that the photodegradation rate depends on the viscosity. This is interpreted as evidence for an intermolecular interaction mechanism. We explore the uncertainty of the estimated photodegradation constant taking into account the uncertainties of the measurements used in the synchronous detection technique.     

Water-Soluble Nanoparticles with Twisted Double [7]Carbohelicene for Lysosome-Targeted Cancer Photodynamic Therapy

Helicene-based therapeutic agents for organelle-targeted photodynamic therapy (PDT) involving both type I and II are challenging and still underexplored. Herein, water-soluble nanoparticles containing twisted double [7]carbohelicene (D7H-NPs) are prepared through self-assembly with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] by a nanoprecipitation method. D7H-NPs display high water solubility with an average size of 46 ± 2 nm. Notably, D7H-NPs can generate efficient singlet oxygen (¹O₂) and superoxide anion (O₂^{· ?}) upon white light irradiation, forming the basis of PDT. Moreover, the typical accumulation in lysosomes of 4T1 cancer cells paves the way to use D7H-NPs for lysosome-targeted cancer phototherapeutics. This paper reports a promising helicene-based phototherapeutic agent involving both type I and II PDT for organelle-targeted biotherapy.     

Nanopatterned monolayers of an adsorbed chromophore

A simple lift-off process was developed to rapidly fabricate nanopatterned photofunctional surfaces. Dye molecules of a perylene derivative (PDID) were adsorbed irreversibly on clean silicon through the holes of an electron-beam lithographied polymer mask. The subsequent removal of the mask in a proper solvent results in PDID nanosized regions of width as small as 30?nm for stripes and of diameter as small as 120?nm for dots. Numerical analyses of atomic force microscopy and laser-scanning confocal microscopy images show that the dye molecules are confined to the regions defined by the lithographic process, with the integrated fluorescence intensity being essentially proportional to the size of the nanofeatures. This demonstrates that a simple organic lift-off process compatible with clean-room technology, and not involving any chemical step, is able to produce photofunctional nanopatterned surfaces, even though the dye is not chemically bonded to the silicon surface.     

Waveguide photodegradation of nonlinear optical organic chromophores in polymeric films

Waveguide photodegradation studies have been carried out to investigate the photostabilities of a series of nonlinear optical chromophores doped into poly(methyl methacrylate) waveguide films. The films were exposed to optical wavelengths lying either within these materials' main absorption bands or in the near-infrared region. Degradation studies were carried out in air, vacuum, and nitrogen environments at room temperature. Experimental results indicate that the principal photodegradation mechanism in operation is photo-oxidation. A simple analytical model indicated the relative sensitivity to photo-oxidation of the materials studied.     

基于CCD器件的苯甲酰脲类农药荧光检测系统

根据苯甲酰脲类农药在紫外光的照射下能够发出荧光的机理,设计了一种基于CCD(电荷耦合器件)的检测该类有机农药残留的光纤荧光测量系统.该系统以脉冲氙灯为激发光源,利用光纤探测和传输荧光,采用线阵CCD代替传统的光电倍增管作为荧光信号的光电检测元件,同时配备A/D高速数据采集卡,实现了单片机控制下荧光信号的光电转换以及数据采集,进而实现了对卡死克和盖虫散(氟铃脲)农药浓度的测量.实验结果表明,在激发波长分别为290nm和345nm时,卡死克和盖虫散的荧光强度分别在360nm和418nm处达到最大,最低检出限分别为12 μg/L和20 μg/L.在25～1 000μg/L范围内,荧光强度和溶液浓度基本呈线性关系.该测量系统灵敏度高,线性范围宽,可以满足荧光检测的要求.     

Quantum dots as enhancers of the efficacy of bacterial lethal photosensitization

Because of the increasing resistance of bacteria to antibiotics there is considerable interest in light-activated antimicrobial agents (LAAAs) as alternatives to antibiotics for treating localized infections. The purpose of this study was to determine whether CdSe/ZnS quantum dots (QD) could enhance the antibacterial activity of the LAAA, toluidine blue O (TBO). Suspensions of Staphylococcus aureus and Streptococcus pyogenes were exposed to white light (3600?lux) and TBO (absorbance maximum = 630?nm) in the presence and absence of 25?nm diameter QD (emission maximum = 627?nm). When the TBO:QD ratio was 2667:1, killing of Staph.?aureus was enhanced by 1.72log(10)?units. In the case of Strep. pyogenes, an enhanced kill of 1.55log(10)?units was achieved using TBO and QD in the ratio 267:1. Singlet oxygen and fluorescence measurements showed that QD suppress the formation of singlet oxygen from TBO and that QD fluorescence is significantly quenched in the presence of TBO (70-90%). Enhanced killing appears to be attributable to a non-F?rster resonance energy transfer mechanism, whereby the QD converts part of the incident light to the absorption maximum for TBO; hence more light energy is harvested, resulting in increased concentrations of bactericidal radicals. QD may, therefore, be useful in improving the efficacy of antimicrobial photodynamic therapy.