Study of Blood Porphyrin Spectral Profile for Diagnosis of Chronic Renal Failure

The progression to end-stage renal failure is independent of the initial pathogenic mechanism. Metabolic acidosis is a common consequence of chronic renal failure that results from inadequate ammonium excretion and decreased tubular bicarbonate reabsorption. Protoporphyrin IX (PpIX) is the immediate metabolic precursor of the heme molecule. The purpose of this study was to evaluate the levels of erythrocytes protoporphyrin IX at an animal model during progressive renal disease. A total of 36 eight-week-old male Wistar rats were divided into six groups: Normal, 4 and 8 weeks after 5/6 nephrectomy (NX). Renal function was evaluated by creatinine clearance and plasma creatinine levels. The autofluorescence of erythrocytes porphyrin of healthy and NX rats was analyzed using fluorescence spectroscopy. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences between normal and NX rats autofluorescence shape occurred in the 600–700 nm spectral region. A correlation was observed between emission band intensity at 635 nm and progression of renal disease.     

无创人皮肤浅表组织血液中卟啉的荧光光谱分析

人体中含有的卟啉化合物主要有血红蛋白、血红素、原卟啉、粪卟啉、尿卟啉等。健康状况影响卟啉代谢,恶性肿瘤影响原卟啉(PPⅨ)与类胡萝卜素的相对含量。采用钛宝石激光器、光导纤维、荧光探头、多色仪、CCD、微机,组建起实施人体皮肤浅表组织内血液中的卟啉进行无创荧光检测系统。对人耳垂皮肤浅表组织内血液进行了荧光分析,结果同P_pⅨ水溶液的荧光谱一致。     

Investigations on photolon-and porphyrin-doped sol-gel fiberoptic coatings for laser-assisted applications in medicine

In view of laser-assisted medical applications, the construction of silica-based sol-gel fiberoptic sensors based on photolon (Ph) and protoporphyrin IX (PP IX) is discussed. Electron microscopy and AFM were used to characterize the silica sol-gel coatings. AFM measurements indicate a change in the surface porosity. The PP IX-based sensors were constructed as a one-layer optode as well as a multilayered structure. An additional hybrid sensor made up of alternate layers of PP IX-and Ph-doped sol-gel was also constructed and examined. Sol-gel matrices were prepared from silicate precursor tetraethylorthosilicate (TEOS) mixed with ethanol in acid-catalyzed hydrolysis. The carrier matrices of photosensitive dyes were produced with factor R = 20, where R denotes the ratio of solvent moles (ethanol) to the number of TEOS moles. A multilayered coating was built up using the reverse-dipping technique. The overall coating thickness was determined by electron microscopy. Doped sol-gels with different PP IX concentrations were used to produce fiberoptic coatings. The film optodes with a different number of layers were examined by fluorescence spectroscopy. It was found that photolon and protoporphyrin IX entrapped in sol-gel preserve their chemical reactivity and have contact with the external environment. The hybrid sensor demonstrated clear fluorescence and a reversible behavior in gaseous environments.     

卟啉和金属卟啉的荧光寿命

在单光子计数仪器上测量了一系列卟啉自由碱和金属卟啉的荧光寿命.在光动力治癌疗法中用作光敏剂的HPD缓冲溶液呈现双指数荧光褒减.τ=3ns的短寿命组分是聚集态HPD的表征,而τ=14ns的长寿命组分是单体HPD的表征,HP缓冲溶液却呈现单指数衰减.有七个卟啉或其金属络合物的寿命是初次报导.某些卟啉化合物的实测寿命与计算值符合良好.     

无长链天然卟啉的LB膜制备与光谱研究

本工作对无长链天然血卟啉(HP),原叶啉(PP)和卟啉C行进了LB膜的成膜探索,制备了单层、多层以及与硬脂酸、卵磷脂混合后的复合LB膜.对它们进行了紫外——可见吸收光谱、荧光光谱和时间分辨荧光光谱的研究,发现在LB膜中荧光寿命是一个很好的表征手段.     

Picosecond time resolved fluorescence spectroscopy study of aggregation of porphyrin derivative Mn(3,4,5-OMeTPP)Cl

The picosecond time-resolved fluorescence spectrum I(, t) of the porphyrin derivative Mn(3,4,5-OMeTPP)Cl is measured. Experiments show that the fluorescence relaxation of the concentrated sample solution is composed of a fast (ps), a moderately fast (100 ps) and a slow (ns) decay process. Furthermore, the fluorescence spectra for various times after excitation have different shapes. Detailed analysis shows that the fluorescence is composed of three components with different relaxation times and spectrum shapes. The three components are attributed to monomers, dimers, and aggregates respectively.     

Fluorescence of the <Emphasis Type="Italic">cis</Emphasis>-dimers of porphyrins containing ethylene bridges

The absorption and fluorescence spectra of solutions of the porphyrin dimers containing ethylene bridges, binding two Zn(II)octaethylporphyrin molecules or one Zn(II)octaethylporphyrin and one octaethylporphyrin molecule into cis conformation, were studied. For both studied cis-dimers, structureless absorption spectra were observed, only in general resembling the spectra of initial molecules, as well as weak fluorescence with a wide bell-shaped spectrum with a maximum at 680–700 nm and a quantum yield of about Φ_f≈3×10^?5, which is three orders of magnitude lower as compared to the fluorescence of initial monomeric porphyrins. A strong dependence of the fluorescence yield on a medium viscosity was revealed. In addition, low-yield irreversible cis-trans photoisomerization of cis-dimers was detected. A mechanism responsible for the strong intramolecular quenching of fluorescence of the porphyrin dimers containing ethylene bridges is suggested to be a conformation transformation in the region of the ethylene bridge, leading the molecular system to the region of “conic crossing” of the energy surfaces of excited and ground electron states.     

Study of ProtoPorphyrin IX Elimination by Body Excreta: A new Noninvasive Cancer Diagnostic Method?

This paper describes the elimination of porphyrins by feces. It was demonstrated that porphyrin accumulates substantially more in tumors than in normal tissues, and consequently more PPIX reaches the blood of patients and animals with tumors, and then, it needs to be eliminated. The fluorescence of feces revealed that there are large amounts of PPIX in the excreta of animals with cancer comparing with healthy animals. The autofluorescence of feces porphyrin extracted with acetone was analyzed using fluorescence spectroscopy of animals inoculated with DU145 cells into the prostate and healthy animals to monitor the PPIX concentration. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences were observed in autofluorescence intensities measured in the 575–725 nm spectral regions for the studied groups. The results showed a noninvasive, simple, rapid and sensitive method to detect cancer by feces analysis.     

Drop‐coating deposition Raman spectroscopy of porphyrins

We report an improvement of the Raman detection sensitivity of porphyrins by factor of 10⁵ using drop‐coating deposition Raman spectroscopy (DCDR). We were able to obtain Raman spectra from ~20 nM initial (deposited) concentrations of cationic and anionic porphyrin. This detection limit is comparable with or even better than that of surface‐enhanced Raman scattering spectroscopy using Ag nanoparticles. Moreover, DCDR spectrum of protoporphyrin IX, which is known as a marker in clinical diagnostics of cancer, was also obtained from 10 nM deposited concentration. To our knowledge, it is the first demonstration of the resonance DCDR spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Spectroscopy of Redox Phototransformations of Porphyrins in Frozen Solutions (Review)

The results of spectral investigations of photochemical transformations of porphyrins and phthalocyanins in frozen solutions have been systematized and critically considered. We investigated the processes responsible for the formation of the products of the primary stages in phototransformations of porphyrins, that is, cations and anions of porphyrins formed in phototransfer of the electron. Photosensitized destruction of the molecules of a frozen matrix with removal of the H atom and its addition to porphyrin leads to the formation of a porphyrin monohydroderivative. In frozen acid media, protonated forms of porphyrins are similar in their photochemical properties to metalloporphyrins: they detach and add electrons, form mono and dihydroderivatives. The process of formation and the properties of the ionicmolecular complexes of Znporphyrin with anions of pyridine, quinoline, and phenazine and also of complexes of the ion–ion type of Znporphyrin anions with pyridine anions have been considered. Owing to the intracomplex charge phototransfer, these formations possess high photochemical activity, which made it possible to use them for solving problems in the field of holeburning spectroscopy.     

基于小波变换的人血清血卟啉荧光光谱分析法

卟啉是生命活动的重要物质,原卟啉可做为肿瘤血液标志物.人血清中血卟啉含量很低,且受多种因素影响.基于小波变换的人血清血卟啉荧光光谱分析法可实现对其弱信号的分辨.用多功能光谱测量系统获取人血清血卟啉荧光光谱,对获取的荧光光谱通过数次小波变换进行分解,将噪声与光谱信号分离,滤波后的荧光光谱进行6次分解后得到了离散逼近信号(a1～a6)和离散细节信号(d1～d6).信号频率随着分解次数的增加逐步降低.当分解到第6次时,出现了血卟啉荧光特征峰,成功地观测到血卟啉的荧光发射峰,实现了弱信号分辨,得到了血清中所含血卟啉的准确成份和定量信息,从而进行定性和定量分析,为血清荧光光谱用于肿瘤的早期诊断提供了一种方法.     

Manifestation of Peripheral NO2-Substitution in the Resonance Raman Scattering Spectra of Tetraarylporphyrins

The resonance Raman scattering (RS) spectra of a series of nitro-substituted derivatives of the free base of tetraphenyl porphyrin that were obtained on continuous excitation near the Soret band have been investigated. For the molecules in which the NO₂ group is located in the -position of pyrrole rings or in the para-position of meso-phenyl substituents, an intense line of the mode within the range 1340–1355 cm^–1 is discovered. Based on the obtained resonance RS spectra of the diprotonated derivatives of nitro-tetraarylporphyrins, it is assigned to symmetrical vibration of a nitro group ( _s NO₂). Activation of the _s NO₂ vibration is indicative of the existence of the -delocalization effect for a number of nitro-substituted porphyrins at which interaction between the molecular orbitals of the nitro-substituents and the porphyrin macrocycle occurs. Moreover, the -delocalization is typical of both direct addition of a nitro-substituent to the porphyrin ring and of its binding via a meso-phenyl group. The results obtained demonstrate the informative value of the resonance RS spectroscopy for creating and studying model donor–acceptor systems with the participation of porphyrins.     

Fluorescence lifetime imaging with a low-repetition-rate passively mode-locked diode-pumped Nd:YVO4 oscillator

We report a wide-field fluorescence lifetime imaging microscope based on a low-repetition-rate (3.7-MHz) passively mode-locked diode-pumped laser source. This inexpensive and compact laser source operating in the visible and UV range can excite a wide range of fluorophores of biological interest. We demonstrate that the power of this laser source is highly sufficient for studying biological systems with low quantum yields (autofluorescence of tissues and stained living cells). The maximum measurable lifetime is also strongly increased with this laser source, as fluorescence intensity measurement can occur 250 ns after the excitation pulse.     

人血清血卟啉荧光光谱的双正交样条小波识别

人血清中血卟啉含量很低,其荧光光谱强度很弱,往往呈肩峰形态,难以识别其位置和荧光强度。双正交样条小波可实现对其弱信号的分辨。在血清背景荧光光谱上叠加血卟啉荧光信号,并改变其荧光强度,得到一系列强度改变的 血清血卟啉荧光光谱。用双正交样条小波bior5.5对获取的荧光光谱通过7次小波分解,将背景与卟啉荧光信号分离,得到了离散逼近信号(a₁～a₇)和离散细节信号(d₁～d₇)。结果 表明,信号频率随着分解次数的增加逐步降低。当分解到第7次时,出现了血卟啉荧光特征峰。信号峰的位置随着信号强度的下降蓝移约2.5 nm,而通过小波滤波器提取的信号峰的位置不变。随着信号的强度降低,信号峰的位置逐渐消失 ,因而其荧光强度与荧光峰的位置无法确定,而通过小波滤波器提取的信号则不受影响,且灵敏度高,从而实现了用双正交样条小波对人血清血卟啉荧光光谱的识别。由于小波变换是线性变换,离散细节信号保持信号原有的线性关系,可 以用来对血清中所含血卟啉的准确成份和定量信息,从而进行定性和定量分析,为血清荧光光谱用于肿瘤的早期诊断提供了一种方法。     

Optical and photophysical investigation of Meso, Proto and Hematoporphyrin(IX)dimethylester doped hybrid borate glasses

Hybrid borate glasses containing different concentrations (0.5-2.0 mg in 12 g of boric acid) of Mesoporphyrin(IX)dimethylester, Protoporphyrin(IX)dimethylester and Hematoporphyrin(IX)dimethylester were prepared by rapid melt quench technique at 230 °C. The hybrid glass samples were characterized by X-ray diffraction, optical absorption, steady state and time-resolved fluorescence emission. The optical absorption spectrum shows red-shift in Soret band along with change in Q-band pattern. The intensity of Q-band was found to increase with increase in the concentration of porphyrin in the glass. Steady state emission spectrum shows strong S₂→S₀ emission in the range 462-495 nm and blue shift in S₁→S₀ emission. Time-resolved fluorescence emission and fluorescence excitation spectra showed that different structures of porphyrins were exist in the glass samples. The variation in the spectral behaviour in the glass was correlated with those in solution medium and possible structures of porphyrin in borate glass were explored.     

用时间分辨光谱研究红细胞悬液的荧光发射

实验获得了激光照射红细胞悬液的荧光光谱,并分别监测不同荧光峰值波长处强度随时间的衰变过程,测试了其相应的荧光寿命。结果表明,在波长为407nm的激光照射下,红细胞悬液向外发射中心波长分别位于596,628,692nm的荧光光谱,各荧光峰对应衰变过程的平均荧光寿命分别为1.97,13.31,14.58ns。利用荧光强度和吸收率的加和性表示了混合物的总吸收率和总荧光发射强度,通过理论计算获得了红细胞悬液中锌卟啉、原卟啉和其他游离物参与荧光发射的相对含量和相对强度在不同荧光峰位的变化关系,进一步解释了不同峰位处荧光发射强度和平均荧光寿命的变化原因。     

Steady state and time-resolved autofluorescence studies of human colonic tissues

Steady state and time-resolved autofluorescence spectroscopies are employed to study the autofluorescence characteristics of human colonic tissues in vitro. The excitation wavelength varies from 260 to 540 nm, and the corresponding fluorescence emission spectra are acquired from 280 to 800 nm. Significant difference in fluorescence intensity of excitation-emission matrices (EEMs) is observed between normal and tumor colonic tissues. Compared with normal colonic tissue, low nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), and high amino acids and protoporphyrin Ⅸ (PpⅨ) fluorescences characterize high-grade malignant tissue. Moreover, the autofluorescence lifetimes of normal and carcinomatous colonic tissues at 635 nm under 397-nm excitation are about 4.32±0.12 and 18.45±0.05 ns, respectively. The high accumulation of endogenous PpⅨ in colonic cancers is demonstrated in both steady state and time-resolved autofluorescence spectroscopies.     

Development of Low-Cost Instrumentation for Single Point Autofluorescence Lifetime Measurements

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.     

Label-Free Spatial Analysis of Free and Enzyme-Bound NAD(P)H in the Presence of High Concentrations of Melanin

The analysis of autofluorescence, often regarded as undesired noise during the imaging of biological samples, allows label free, unbiased detection of NAD(P)H and melanin in native samples. Because both the emission and absorption spectra of these fluorophores overlap and they can hence not be differentiated using emission filters or with different excitation wavelengths, fluorescence lifetime imaging microscopy (FLIM) is used to differentiate between them. In the present paper the application of two-photon excitation microscopy is presented to investigate the autofluorescence of fungal spores. The model organism which was examined is Aspergillus ochraceus. Furthermore a strategy is developed which allows to quantitatively analyze the fluorescence lifetimes of melanin, free NAD(P)H and protein-bound NAD(P)H using forward convolution of a multiexponential decay function with the instrument response function (IRF) and subsequent fitting to the experimental fluorescence data. As a consequence proteins, which are able to bind NAD(P)H, are located with sub-cellular resolution. Furthermore a spatial differentiation of the fluorophores NAD(P)H and melanin inside the spores, is revealed.     

Spin coupling in distorted high-valent Fe(IV)-porphyrin radical complexes

In order to study structural influences on the interaction of Fe(IV) (S=1) and porphyrin cation radical (S=1/2) in high-valent iron porphyrin complexes of the type ¦X-(TMP)Fe=O¦⁺(Cl^–), X=I, Br₂, Br₄ were generated by mCPBA oxidation of corresponding Fe(III) porphyrins. The halogen substitution at the peripheral positions of the porphyrin leads to distortion of the planar porphyrin ring of ¦(TMP)Fe=O¦⁺. The new species have beeen investigated by temperature-dependent EPR and field-dependent Mössbauer spectroscopy; for the evaluation of spectra, we adopted the spin-Hamiltonian formalism including exchange interaction explicitly. As in ¦(TMP)Fe=O¦⁺, strong ferromagnetic spin coupling was observed with|J₀|D=0.9–1 and a zero-field spltting ofD32 cm^–1. For consistent parametrization of EPR and Mössbauer results, anisotropic coupling had to be introduced. Compared to ¦(TMP)Fe=O¦⁺ [1], analysis of the spectroscopic data shows that zero-field splitting and spin coupling is only slightly affected by the halogen distortion of the porphyrin structure.