Fluorescence of a photosensitizer based on an indotricarbocyanine dye in photochemotherapy

We present the results for studies of the spectral luminescence properties of a symmetric indotricarbocyanine dye (PD1) in HeLa tumor cells and animal tissues in vivo during a photochemotherapy session and after the end of the session. We have established that when the dye is exposed to light in tumor tissues, changes occur in the position and half-width of the dye fluorescence spectra, while in a culture of HeLa cells its spectral characteristics are constant. Based on analysis of the effect of overlap between the absorption spectra of endogenous biomolecules and the fluorescence spectra of the dye plus comparison of the experimental data with numerical modeling results, we have concluded that the observed changes in the fluorescence spectra of PD1 in vivo are due to a change in the ratio of the different forms of hemoglobin in the tumor tissue. We have shown that the spectral characteristics of PD1, fluorescing in the near IR range, correlate with the depth of tumor tissue necrosis achieved on exposure to light. We have established that tumor tissue necrosis occurs down to a depth of 2 cm in the case of all strains studied: S-45, SM-1, and W-256, where as a result of exposure to light, we observe an increase in the half-width and a short-wavelength shift of the fluorescence spectrum of the dye PD1, and also the intensity of its fluorescence does not recover.     

Fluorescence of the Polymethine Dye Tiks and Diagnostics af Cancer

It is shown that the fluorescence of the polymethine dye TIKS, whose absorption and fluorescence bands are located in the spectral region of transmission of biological tissues, can be recorded from a depth of up to 1.5 cm of an animal's body. The intensity of the fluorescence recorded from the surface of the animal's body in intravenous injection of the dye (1–2 mg/kg) is in direct proportion to its concentration in tumor nodes and muscles. In rapidly growing tumors, a high (up to 3.6) degree of contrast of the content of the dye is attained in tumor tissues as compared to the surrounding normal tissues. Over the course of 7 days after the injection, the dye is practically completely removed from both the tumor and normal muscular tissues. From the change in the fluorescence intensity in scanning the surface one can determine the regions of localization of tumor nodes against the background of the surrounding normal tissues and the presence of regions with a nonuniform distribution of the dye.     

Fluorescence spectra of blood plasma treated with ultraviolet irradiation <Emphasis Type="Italic">in vivo</Emphasis>

We have studied the fluorescence spectra of blood plasma from patients with acute coronary syndrome, and also the effect of therapeutic doses of in vivo ultraviolet blood irradiation (UBI) on the spectra. We have established that the maxima in the fluorescence spectra of the original plasma samples, obtained from unirradiated blood, are located in the wavelength interval 330–340 nm, characteristic for the fluorescence of tryptophan residues. In extracorporeal UBI (λ = 254 nm), we observed changes in the shape and also both a blue and a red shift in the maxima of the fluorescence spectra, differing in magnitude for blood plasma samples from different patients in the test group. We show that UBI-initiated changes in the fluorescence spectra of the plasma depend on the original pathological disturbances of metabolite levels, and also on the change in the oxygen-transport function of the blood and the acid–base balance, affecting the oxidative stability of the plasma. We have concluded that UV irradiation, activating buffer systems in the blood, has an effect on the universal and specific interactions of the tryptophan residue with the amino acid residues and water surrounding it.     

Determination of photosensitizer concentration in biological tissues from diffuse reflectance and fluorescence

We present a fiber-optic device for noninvasive determination of the optical properties of biological tissues and photosensitizer concentration. The device developed can be used in two modes: the mode for detection of the spatial distribution of diffuse reflectance, and the fluorescence mode. We tested the device in vitro on specimens simulating tissue and on whole blood, and also in vivo on rats. We have shown that with additional modification and adaptation, the device can be used for in vivo monitoring of parameters important for photodynamic therapy.     

Wavelength-dependent rotation of dye molecules in a polar solution

Investigation of rotation movement of 3-amino-N-methylphthalimide in glycerol was carried out, taking into consideration the fluctuation of solvate structure. It was shown theoretically and experimentally that structural relaxation of the solvate shell, which follows excitation of the dye molecule, causes not only shift of the fluorescence spectrum in time but also additional rotation of the dye molecule. This effect, which may be called wavelength-dependent rotation, depends on the light frequency of both excitation and fluorescence. In particular, at excitation near the maximum of the absorption band, when the relaxation process is followed with the red shift of the fluorescence maximum, the anisotropy of fluorescence decreases faster in the red part of the fluorescence band than in the blue part. On the contrary, in the case of far anti-Stokes excitation, when the temporal shift of fluorescence is going to the blue, the anisotropy in the red part of the spectrum drops more slowly than in the blue part. Finally, there is a special excitation frequency which causes neither change of the fluorescence maximum nor acceleration of the rotational movement of the dye molecule. It is also shown that the temporal evolution of the spectrum and anisotropy of fluorescence in a polar dye solution may be quantitatively described using the socalled inhomogeneous broadening function (IBF). This function gives the distribution of dye molecules in a solution over frequencies of pure electronic transition due to fluctuations of the surrounding shell structure. Measurements of IBF changes in time carried out for 3-amino-N-methylphthalimide showed that during first 3 ns after excitation, the half-width of the IBF grows, and at the same time its maximum quickly shifts to the red. At the later time period there are only small changes of IBF position but considerable exponential decrease in its half-width. The IBF during this period preserves the Gaussian shape.     

荧光光谱法检测角膜晚期糖基化终末产物研究

晚期糖基化终末产物（AGEs）是一种结构多样的化合物,在人体血糖高于正常范围时,会大量产生且不能通过自身代谢降解,具有血糖长期异常的记忆作用。研究表明AGEs是引起糖尿病及其并发症的重要因素之一,通过检测体内AGEs的积累情况可以预测糖尿病及其并发症的发生和发展进程。现有的离体AGEs检测方法存在操作复杂、时间较长、成本较高和不易推广等问题;在体AGEs检测方法存在皮肤色素、年龄和血红蛋白干扰等问题。为此,基于角膜良好的光学特性和AGEs的自体荧光特性,提出一种角膜晚期糖基化终末产物荧光光谱检测方法。构建了一套角膜AGEs荧光光谱检测系统,系统由微型光纤光谱仪、集成LED激发光源、Y型12+1光纤和PC端光谱处理显示软件组成。荧光光谱检测系统采用激发光源波长分别为370和395 nm在暗室条件下对17名志愿者（男性9人,女性8人,糖尿病患者4人,年龄最小15周岁,最大81周岁）进行数据采集,得到激发光波长分别为370和395 nm的荧光光谱数据。为了准确识别荧光光谱中的有用信息,先截取需要的荧光光谱数据段（450~700 nm）,然后对其进行去除背景噪声、归一化、小波变化等方法处理,可以将荧光光谱中不明显的荧光峰值进行放大和识别。实验结果发现,采用波长为370和395 nm的LED作为激发光源,检测到角膜发射的荧光光谱范围在420~600 nm内,并且都分别在450~500,500~550和550~600 nm三个范围内存在光谱峰值。根据荧光性物质的荧光峰值与激发光波长无关的原理,表明两种不同波长的激发光所得到的荧光光谱都是由同一种物质AGEs产生。对糖尿病患者和正常人的荧光峰值强度进行分析,显示糖尿病患者的荧光强度明显高于正常人,表明本研究通过荧光光谱法检测角膜晚期糖基化终末产物具有可行性。     

On the Membrane Binding of the Potentiometric Probe Di-4-ANEPPS: A Fluorescence and Resonance Raman Study

Di-4-ANEPPS is a dye molecules which has been used to probe membrane electric potentials by its electrochromic response. The dye has an unusual blue shift in the absorption spectrum upon partitioning to lipid membranes. We studied its binding properties to membranes by absorption, fluorescence and resonance Raman spectroscopy. The latter technique provided evidence for the differential solubilization of the dye in the membrane, where the polar head of the molecule protrudes out of the lipid environment and is responsible for the spectral behavior.     

Spectral and temporal characteristics of fluorescence of drops comprising dissolved dye excited by the IR laser radiation

Fluorescence of solutions of rhodamine 6G in the form of single drops of millimeter sizes excited by the intense IR and visible laser radiation (with duration of laser pulses of ∼10 ns and wavelengths of 1064 and 532 nm) is experimentally investigated. It was found that the power thresholds of fluorescence excitation for drops comprising the dissolved dye are, by an order of magnitude, less than for a cell with dissolved dye. The dichromatic drop fluorescence has been revealed, which is manifested through the occurrence of two maxima in the fluorescence spectrum of drops comprising dissolved rhodamine 6G. The first maximum coincides with that of the dye fluorescence spectrum, while the second maximum is shifted by ∼1000 cm⁻¹ and is on the slope of the fluorescence line profile. It is shown that the duration of drop fluorescence does not exceed the duration of the exciting pulse.     

Picosecond spectroscopy of intermolecular interactions in dye solutions

Some new experimental data on the time development of ultrashort superfluorescence (SF) in the dye solution are presented. The correlation between temporal behaviour change and the change of the spectral and spatial intensity distribution gives direct evidence of the important role of the selffocusing phenomenon in the formation of the ultrashort SF pulse synchronized with the pumping one. The oriental relaxation of solvent molecules in the dye solution not only influences the duration of the dye SF via the selffocusing mechanism but also causes a temporal change of spontaneous emission spectrum. At least two different relaxation times were found in the fluorescence spectrum of the dye in several polar solutions.     

Influence of the mineral component in biotissues affected by calcinosis on their laser-induced fluorescence spectrum

Laser-induced fluorescence (LIF) spectra of human cardiac-valve tissues affected by calcinosis and of macroscopic calcinosis formations produced from resected cardiac valves with such pathology were investigated by comparison with analogous spectra of bone and myocardial tissue of an animal, LIF was excited by an excimer laser with λ=248 nm. In the spectrum of macroscopic calcinosis formations, luminescence bands of the mineral component were separted from the band of protein tissues when these tissues were subjected to heat treatment at 700°C. The investigations have shown that the LIF spectrum of a calcified tissue contains fluorescence bands of tryptophan and fluorescence bands of the mineral component (hydroxylapatite). A change in the spectrum of tissues with pathological processes can be cause not only by a relative change in the concentration of collagen elastin, but also by the additional fluorescenced of the hydroxylapatite mineral that is absent in a healthy cardiac tissue. It was found that the change in the spectrum characterized by the ratio of the LIF intensity at λ=330 nm to the LIF intensity at λ=450 nm is sufficiently large. This makes it possible to use measurements of the relative intensity of fluorescence at these wavelengths to control the degree of calcination of cardiac tissue. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 66, No. 6, pp. 846–849, November–December, 1999.     

Fluorescence enhancement of N2O2-type dipyrrin ligand in two step responding to zinc(II) ion

Fluorescence imaging is well-suited for the live imaging of biological Zn(II), which has no facile spectroscopic or magnetic signature. The successful application of this methodology requires the development of robust Zn(II) imaging agents that display high sensitivity, selectivity and temporal fidelity. In this study, a N₂O₂-type dipyrrin based bimolecular zinc(II) complex was produced and shown to have sharper, blue-shifted and more enhanced fluorescence emission. An approximate three-fold fluorescence enhancement was achieved within the micromolar concentration range, which is an important parameter for Zn(II) detection in vivo. The increase in emission intensity was due to the dominant role of aryl-ring rotation in governing the excited state dynamics and fluorescence properties of the dipyrrin dye. Fluorescence titration showed that the ligand complex exhibited very strong zinc(II) binding affinity when compared to that in the binuclear chloro complex. The fluorescence emission changes in the dipyrrin dye to zinc(II) ion could be observed not only using instruments but also by the naked-eye (violet→sky blue).     

Fluctuations in synchronously mode-locked dye lasers

We present detailed experimental data on random fluctuations of the pulse properties of a cw rhodamine 6G dye laser synchronously pumped by an acousto-optically mode-locked argon ion laser. It is shown that quantitative information about the fluctuations of the energy, the pulse repetition time and the duration of the pulses can be obtained from the power spectrum of the laser intensity which is measured with the use of an electronic spectrum analyser. This method is capable of revealing small, subpicosecond temporal pulse jitter. We show that the dye laser pulses exhibit an absolute r.m.s. jitter of 20 ps which is induced by the pump laser. The relative jitter in a dual system can be less than 1 ps because well defined correlations of the output fluctuations exist when two lasers are pumped by a common source.     

Relaxation of green fluorescent protein chromophore anion observed by photodissociation in an electrostatic storage ring

The gas-phase absorption properties of the green fluorescent protein chromophore anion were studied using an electrostatic storage ring. The time sequence of neutral particles produced by photodissociation was detected following laser irradiation. The lifetimes of the photo-absorbed ions depended on their storage time in an ion trap before injection into the storage ring. The lifetime increased with the storage time and saturated, indicating a change in the population of rovibrationally excited states with respect to the storage time. Photodissociation neutral spectrum of the relaxed ions measured as a function of the laser wavelength was characterized by a narrow asymmetric shape, which was in good agreement with the photo fragment ion action spectrum reported recently.     

电压敏感染料di-4-ANEPPS的光谱研究

实验研究了电压敏感染料di-4-ANEPPS在家兔心肌组织中的吸收光谱和荧光光谱特性。结果表明,含染料组织的光吸收普遍大于对照组,在450～550 nm波段吸收谱差异更明显;染料在心室组织中的最大吸收峰为(479.75±0.44) nm。通过测量含染料心脏不同部位的荧光光谱,首次发现心室组织、心房组织和主动脉的最大荧光峰位有一定差异,其相对荧光强度则与染料的分布浓度有关。根据三维和二维荧光光谱分别确定了含染料心房和心室组织的最佳荧光激发波长和荧光测定波长。利用心房和心室组织的静息电位差,在不同波长激发光下测量了染料的荧光光谱移动,确定了光标测量实验的最佳激发光和相应荧光检测波长范围。这些研究结果为心脏光学标测系统的设计提供了理论依据。     

Complexation of pyrene and anthracene with human blood plasma

We have studied the interaction between polycyclic aromatic hydrocarbons (pyrene and anthracene) with human serum albumin (HSA) and human blood plasma. We have shown that the increase in the fluorescence intensity and the decrease in the polarity index of pyrene on going from an aqueous solution to a pH 7.4 buffer solution of HSA suggests that polycyclic aromatic hydrocarbons are localized in the hydrophobic microphase of the proteins. The increase in the fluorescence intensity for anthracene and pyrene, and also the decrease in the polarity index of pyrene on going from HSA to blood plasma is connected with the fact that polycyclic aromatic hydrocarbons can bind both to plasma proteins and to plasma lipids. When sodium dodecyl sulfate (SDS) is added to the blood plasma in a concentration greater than the critical micelle concentration, we observe an increase in the fluorescence intensity and the polarity index of pyrene. We hypothesize that this is connected with localization of pyrene near the interface between the hydrophobic and hydrophilic phases of the protein-SDS system. We have established that SDS leads to a change in the structure of blood plasma proteins and promotes escape of polycyclic aromatic hydrocarbons from the protein globules. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 3, pp. 379–382, May–June, 2008.     

基于修正脉搏色素谱的循环血量检测方法

循环血量(CBV)作为主要的血流动力学参数,在心血管疾病的病情评估和手术监护中具有重要的临床应用价值。将吲哚菁绿色素(ICG)作为示踪剂的脉搏色素谱法,通过建立ICG稀释排泄的色素谱曲线,实现CBV的在体无创测量。在实际临床应用中,由于受到血氧波动和环境背景光等干扰因素的影响,脉搏色素谱法测量CBV的准确度低于预期值。为解决这一问题,研究了一种基于修正脉搏色素谱的循环血量检测方法。具体操作是,在患者的肘静脉处注入吲哚菁绿试剂,利用光电传感器分别采集特征波长点的透射光谱信号和背景光电信号,采用差分算法消除血氧波动和环境背景光的干扰影响,建立准确的ICG色素谱曲线,从而计算CBV等血流动力学参数。与131I同位素“金标准法”相比较的试验结果表明,该研究提出的基于修正脉搏色素谱的循环血量检测方法,将CBV测量的平均相对误差从6.85%降低为4.53%,显著提高了其测量准确度。     

Fluorescent Microcrystals Obtained from Coumarin 6 Using the Reprecipitation Method

The reprecipitation method was applied to Coumarin 6. A concentrated solution of the dye in acetone was mixed at room temperature with a large volume of water, and microcrystals rapidly appeared in the medium. Their size and shape were visualized by fluorescence microscopy, as well as by transmission electron microscopy. The absorption and fluorescence properties were studied on the suspensions and compared with those of the dissolved dye. This study is aimed at clarifying the influence of the reprecipitation process upon the fluorescence properties of the dye in the solid state.     

Particular features of incorporation of coumarin 30 into nanoparticles from metal complexes and the intensity of its columinescence

We have studied the sensitized fluorescence of coumarin 30 incorporated into nanoparticles from complexes of p-phenylbenzoyltrifluoroacetone and 1,10-phenanthroline with Y, La, Lu, Gd, Al, and Sc ions in 10% alcoholic-aqueous solutions. We have shown that, upon formation of nanoparticles from complexes of Y(III) and Ln(III) ions, coumarin 30 molecules are completely incorporated from the solution into nanoparticles from complexes up to dye concentrations in the solution comparable with the concentration complexes. For the nanoparticles under study, in the whole range of the examined dye concentrations, concentration quenching of the coumarin 30 cofluorescence has not been observed. Our results show that coumarin 30 is incorporated into lanthanide and yttrium complexes as a synergistic bidentate ligand. The possibility of creating brightly luminescent markers that absorb not only in the range of 360?C370 nm, but also in the range of 440?C450 nm, and have a narrow fluorescence spectrum with ??_max = 520 nm has been demonstrated.     

时间分辨光谱法用于离体人肺鳞癌,腺癌及正常组织的荧光寿命测量

用Ｎｄ：ＹＡＧ倍频光５３２．０ｎｍ泵浦ＤＣＭ染料输出的６３２．８ｎｍ激光作激发源，通过时间分辨法测得离体人肺腺癌，鳞癌及正常组织的特征峰荧光寿命分别为５．１，５．６和６．３ｎｓ实验结果表明：肿瘤组织的荧光寿命总小于正常组织的荧光寿命，这种方法将有可能用于精确诊断和定位肿瘤。     

Photophysics and lasing correlation of pyrromethene 567 dye in crosslinked polymeric networks

The photophysical properties of the pyrromethene 567 dye incorporated in copolymers of methylmethacrylate with different acrylic and methacrylic crosslinking monomers are reported. In general, the solid matrices improve the fluorescence capacity of the dye, due to both an increase and a decrease in the radiative and non-radiative deactivation rate constants, respectively, as consequence of a more rigid environment. It is observed that there is an optimal crosslinking degree for the highest fluorescence efficiency, which depends on the nature of the crosslinking monomer. Taking into account the lasing properties for these systems, it is established a good correlation between the lasing and the fluorescence characteristics of the dye in agreement with previous conclusions obtained in liquid solutions.