Parallel factor analysis of spider fluorophores

Fluorophores from the hemolymph of yellow sac spiders (Cheiracanthium mildei) have been characterized using excitation emission matrix (EEM) fluorescence spectroscopy. This approach provides characterization of fluorophores present in the organism without having to isolate pure samples. Minimal variation occurs between individual samples and each EEM has two distinct peaks, suggesting two fluorophores may be present in the hemolymph. Parallel factor analysis reveals that three fluorophores (with excitation and emission maxima at 270/319, 330/389, and 350/465 nm) best explains the sample to sample variation. By comparing the spectra of the three individual components to fluorophores found in scorpions it is shown that these spiders possess different fluorophores than scorpions. Furthermore, the fluorescence observed is not consistent with beta-carboline or 4-methyl-7-hydroxycoumarin, two compounds previously described in scorpions.     

Diagnosis of early stage nasopharyngeal carcinoma using ultraviolet autofluorescence excitation-emission matrix spectroscopy and parallel factor analysis

We report the diagnostic ability of ultraviolet (UV)-excited autofluorescence (AF) excitation-emission matrix (EEM) spectroscopy associated with parallel factor (PARAFAC) analysis for differentiating cancer from normal nasopharyngeal tissue. A bifurcated fiber-optic probe coupled with an EEM system was used to acquire tissue AF EEMs using excitation wavelengths between 260 and 400 nm, and emission collection between 280 and 500 nm. A total of 152 AF EEM landscapes were acquired from 13 normal and 16 nasopharyngeal carcinoma (NPC) thawed ex vivo tissue samples from 23 patients. PARAFAC was introduced for curve resolution of individual AF EEM landscapes associated with the endogenous tissue constituents. The significant factors were further fed to a support vector machine (SVM) and cross-validated to construct diagnostic algorithms. Both the EEM intensity landscapes and the PARAFAC model revealed tryptophan, collagen, and elastin to be the three major endogenous fluorophores responsible for the AF signal from normal and NPC tissues. The EEM intensity distribution and PARAFAC factors suggest an increase of tryptophan and a decrease of collagen and elastin in NPC tissues compared to the normal. The classification results obtained from the PARAFAC-SVM modeling yielded a diagnostic accuracy of 94.7% (sensitivity of 95.0% (76/80); specificity of 94.4% (68/72)) for normal and NPC tissue differentiation. This study suggests that UV-excited AF EEM spectroscopy integrated with PARAFAC algorithms has the potential to provide clinical diagnostics of early onset and progression of NPC.     

Spectroscopic diagnosis of colonic dysplasia.

We have developed a method for defining diagnostic algorithms for pathologic conditions based on fluorescence spectroscopy. We apply this method to human colon tissue and show that fluorescence can be used to diagnose the presence or absence of colonic adenoma. This method uses fluorescence excitation-emission matrices (EEM) to identify optimal excitation regions for obtaining fluorescence emission spectra which can be used to differentiate normal and pathologic tissues. In the case of normal and adenomatous colon tissue, these were found to be: 330, 370, and 430 nm +/- 10 nm. At these excitation wavelengths, emission wavelengths for use in diagnostic algorithms are identified from average difference and ratio of the spectra from normal and pathologic tissues. In colon tissue, at 370 nm excitation, 404, 480, and 680 nm were found to be useful emission wavelengths for diagnosing the presence of adenoma in vitro. The basis of colon tissue autofluorescence was investigated using EEM of pure molecules and relevant excitation-emission maxima in the literature.     

Spectroscopic studies on the interaction between riboflavin and albumins

The interactions between riboflavin (RF) and human and bovine serum albumin (HSA and BSA) were studied by using absorption and fluorescence spectroscopic methods. Intrinsic fluorescence emission spectra of serum albumin in the presence of RF show that the endogenous photosensitizer acts as a quencher. The decrease of fluorescence intensity at about 350 nm is attributed to changes in the environment of the protein fluorophores caused by the ligand. The quenching mechanisms of albumins by RF were discussed. The binding constants and binding site number were obtained at various temperatures. The distance between albumins and RF in the complexes suggests that the primary binding site for RF is close to tryptophan residue (Trp214) of HSA and Trp212 of BSA. The hydration process of albumins has also been discussed.     

Multivariate curve resolution analysis excitation-emission matrices of fluorescence of humic substances

Excitation-emission matrices (EEM) of fluorescence of aqueous solutions of humic substances (HS), and sets of EEM acquired as function of the HS concentration, were analysed by multivariate curve resolution alternating least squares (MCR-ALS). Three types of HS samples were studied: one commercial humic acid; two samples of fulvic acid (FA) extracted from a pinewood soil; two samples of FA extracted from recycled wastes. The fluorescence measurements were carried out at HS concentration between 5 and 100 mg/L and at pH 6. The application of MCR-ALS algorithm on each individual EEM, as well as on column-wise augmented matrices, allows the identification of three major fluorophores in all HS samples analysed. The emission and excitation spectra of these fluorophores were recovered and are characteristic of each sample. Moreover, the variation of the fluorescence intensities of each fluorophore with HS concentration shows deviations from linearity at HS concentration higher than 30 mg/L, depending on the fluorophore and/or sample. This behaviour reveals the existence of inner filter effects that affect the proportionally between the fluorescent signal and concentration but do not provoke measurable distortions on the fluorescence spectra of the detected fluorophores.     

Characterization of the autofluorescence of polymorphonuclear leukocytes, mononuclear leukocytes and cervical epithelial cancer cells for improved spectroscopic discrimination of inflammation from dysplasia

Fluorescence spectroscopy has the potential to improve the in vivo detection of intraepithelial neoplasias; however, the presence of inflammation can sometimes result in misclassifications. Inflammation is a common and important pathologic condition of epithelial tissues that can exist alone or in combination with neoplasia. It has not only been associated with the presence of cancer but also with the initiation of cancer by damage induced due to the oxidative activity of inflammatory cells. Microscopic examination of cervical biopsies has shown increased numbers of polymorphonuclear and mononuclear leukocytes in inflamed tissues mostly confined to the stroma. The purpose of this study was to characterize the fluorescence properties of human polymorpho- and mononuclear leukocytes and compare their fluorescence to that of cervical cancer cells. Human neutrophils were purified from peripheral blood and their fluorescence characterized over an excitation range of 250-550 nm. There are four notable excitation emission maxima: the tryptophan peak at 290 nm excitation, 330 nm emission; the NAD(P)H peak at 350 nm excitation, 450 nm emission, the FAD peak at 450 nm excitation, 530 nm emission and an unidentified peak at 500 nm excitation, 530 nm emission. Treatment of these peripheral blood neutrophils with 40 nM phorbol myristate acetate or with the chemotactic peptide formyl-Met-Leu Phe (1 M) demonstrated a significant increase in NAD(P)H fluorescence. Isolated mononuclear cells have similar emission peaks for tryptophan and NAD(P)H and a small broad peak at 450 nm excitation, 530 nm emission suggestive of FAD. Comparison of the fluorescence from leukocytes to epithelial cancer cell fluorescence has demonstrated the presence of these fluorophores in different quantities per cell. The most notable difference is the high level of tryptophan in cervical epithelial cancer cells, thus offering the potential for discrimination of inflammation.     

基于萘酰亚胺的生物硫醇探针的合成及对含硫醇氨基酸的检测

合成了以4-羟基萘酰亚胺为荧光团,2,4-二硝基苯磺酰氧基为特异性识别基团的生物硫醇探针4-(2,4-二硝基苯磺酰氧基)-正丁基-1,8-萘酰亚胺(DNSBN).吸收光谱和荧光光谱结果表明, DNSBN对半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)3种生物硫醇分子具有高效的检测识别能力,不受其它17种天然氨基酸的干扰.同时,通过荧光滴定实验证实了此探针是一种比率型探针,555 nm处的荧光强度与溶液中的生物硫醇分子浓度在0 ~ 20 μmol/L范围内呈良好的线性关系,对Cys、Hcy和GSH的检出限(3σ)分别为25.9、92.0和77.9 nmol/L.而吸收光谱、荧光光谱和质谱表征数据显示,生物硫醇与2,4-二硝基苯磺酸酯发生亲核取代反应并导致磺酸酯的分解.随着识别基团的解离,探针分子的d-PeT (donor-excited photoinduced electron transfer) 效应被解除,并出现非常明显的比色与荧光变化.HeLa细胞成像实验表明,探针DNSBN具有良好的生物相容性,能够对细胞外源性生物硫醇分子进行检测.     

Synthesis of new two-photon absorbing fluorene derivatives via Cu-mediated Ullmann condensations

The Ullmann amination reaction was utilized to provide access to a number of fluorene analogues from common intermediates, via facile functionalization at positions 2, 7, and 9 of the fluorene ring. Through variation of amine or iodofluorene derivative, analogues bearing substitutents with varying electron-donating and electron-withdrawing ability, e.g., diphenylamino, bis-(4-methoxyphenyl)amine, nitro, and benzothiazole, were synthesized in good yield. The novel fluorene derivatives were fully characterized, including absorption and emission spectra. Didecylation at the 9-position afforded remarkably soluble derivatives. Target compounds 4, 5, and 9 are potentially useful as fluorophores in two-photon fluorescence microscopy. Their UV-vis spectra display desirable absorption in the range of interest suitable for two-photon excitation by near-IR femtosecond lasers. Preliminary measurements of two-photon absorption indicate the derivatives exhibit high two-photon absorptivity, affirming their potential as two-photon fluorophores. For example, using a 1,210 nm femtosecond pump beam, diphenylaminobenzothiazolylfluorene 4 exhibited nondegenerate two-photon absorption, with two-photon absorptivity (delta) of ca. 820 x 10(-50) cm(4) s photon(-1) molecule(-1) at the femtosecond white light continuum probe wavelength of 615 nm.     

食品中苏丹红Ⅰ和辣椒红的表面增强拉曼散射(SERS)光谱与激发-发射矩阵荧光光谱鉴别

利用表面增强拉曼散射(SERS)光谱及激发-发射矩阵(EEM)荧光光谱对食品中非法添加剂苏丹红Ⅰ和合法食品添加剂辣椒红进行了定性分析和检测. SERS光谱结果表明, 苏丹红Ⅰ在低波数区域分子的扭转振动信号增强比较明显; 而辣椒红在1521和1158 cm^-1处拉曼信号增强效果比较明显; EEM荧光光谱结果表明, 苏丹红Ⅰ的乙醇溶液在P₁(λ_ex=285 nm, λ_em=345 nm)和P₂(λ_ex= 335 nm, λ_em=548 nm)处有2个明显的荧光特征峰; 而辣椒红的乙醇溶液有3个特征荧光峰, 分别为P₁(λ_ex=545 nm, λ_em=580 nm), P₂(λ_ex=560 nm, λ_em=665 nm)和P₃(λ_ex=608 nm, λ_em=672 nm).     

Optimal excitation wavelengths for in vivo detection of oral neoplasia using fluorescence spectroscopy

There is no satisfactory mechanism to detect premalignant lesions in the upper aero-digestive tract. Fluorescence spectroscopy has potential to bridge the gap between clinical examination and invasive biopsy; however, optimal excitation wavelengths have not yet been determined. The goals of this study were to determine optimal excitation-emission wavelength combinations to discriminate normal and precancerous/cancerous tissue, and estimate the performance of algorithms based on fluorescence. Fluorescence excitation-emission matrices (EEM) were measured in vivo from 62 sites in nine normal volunteers and 11 patients with a known or suspected premalignant or malignant oral cavity lesion. Using these data as a training set, algorithms were developed based on combinations of emission spectra at various excitation wavelengths to determine which excitation wavelengths contained the most diagnostic information. A second validation set of fluorescence EEM was measured in vivo from 281 sites in 56 normal volunteers and three patients with a known or suspected premalignant or malignant oral cavity lesion. Algorithms developed in the training set were applied without change to data from the validation set to obtain an unbiased estimate of algorithm performance. Optimal excitation wavelengths for detection of oral neoplasia were 350, 380 and 400 nm. Using only a single emission wavelength of 472 nm, and 350 and 400 nm excitation, algorithm performance in the training set was 90% sensitivity and 88% specificity and in the validation set was 100% sensitivity, 98% specificity. These results suggest that fluorescence spectroscopy can provide a simple, objective tool to improve in vivo identification of oral cavity neoplasia.     

Functional Near‐Infrared Fluorescent Probes

Near‐infrared (NIR) fluorescent probes have attracted much attention, but despite the availability of various NIR fluorophores, only a few functional NIR probes, that is, probes whose absorption and/or fluorescence spectra change upon specific reaction with biomolecules, have been developed. However, functional probes operating in the NIR range that can be targeted to protons, metal ions, nitric oxide, β‐galactosidase, and cellular stress markers are expected to be effective for fluorescence imaging in vivo. This Focus Review concentrates on these functional NIR probes themselves, not their applications.     

FLUORESCENCE OF MELANIN-DEPENDENCE UPON EXCITATION WAVELENGTH AND CONCENTRATION

Abstract— An introduction to the fundamental characteristics of synthetic melanin fluorescence is presented. The particular difficulties associated with the detection and reduction of the relatively weak signal are discussed and a technique is described for correcting the fluorescence spectra for attenuation of the excitation and emission beams. Spectra are reported for the excitation wavelength range 340–400 nm and an emission range of 360–560 nm. The concentration dependence of the corrected fluorescence signal is examined and is shown to be linear. The variation of the fluorescence spectra with excitation wavelength suggests a two-component fluorescence, for the wavelength range studied. The presence of an isosbestic point in the spectra is used to identify the fluorophores as components of a reaction equilibrium. The possible relationship of this equilibrium to that associated with the melanin photo ESR is discussed     

Generation of DNA photolesions by two-photon absorption of a frequency-doubled Ti:sapphire laser

The formation of spatially localized regions of DNA damage by multiphoton absorption of light is an attractive tool for investigating DNA repair. Although this method has been applied in cells, little information is available about the formation of lesions by multiphoton absorption in the absence of exogenous or endogenous sensitizing agents. Therefore, we have investigated DNA damage induced in vitro by direct two-photon absorption of frequency-doubled femtosecond pulses from a Ti:sapphire laser. We first developed a quantitative polymerase chain reaction assay to measure DNA damage, and determined that the quantum yield of lesions formed by one-photon absorption of 254 nm light is 7.86×10(-4). We then measured the yield of lesions resulting from exposure to the visible femtosecond laser pulses, which exhibited a quadratic intensity dependence. The two-photon absorption cross section of DNA has a value (per nucleotide) of 2.6 GM at 425 nm, 2.4 GM at 450 nm, and 1.9 GM at 475 nm. A comparison of these in vitro results to several in vivo studies of multiphoton photodamage indicates that the onset of DNA damage occurs at lower intensities in vivo; we suggest possible explanations for this discrepancy.     

Differential Laser‐Induced Perturbation Spectroscopy for Analysis of Mixtures of the Fluorophores l‐Phenylalanine,l‐Tyrosine and l‐Tryptophan Using a Fluorescence Probe

Quantitative detection of common endogenous fluorophores is accomplished using differential laser‐induced perturbation spectroscopy (DLIPS) with a 193‐nm UV fluorescence probe and various UV perturbation wavelengths. In this study, DLIPS is explored as an alternative to traditional fluorescence spectroscopy alone, with a goal of exploring natural fluorophores pursuant to biological samples and tissue analysis. To this end, aromatic amino acids, namely, l ‐phenylalanine, l ‐tyrosine and l ‐tryptophan are mixed with differing mass ratios and then classified with various DLIPS schemes. Classification with a traditional fluorescence probe is used as a benchmark. The results show a 20% improvement in classification performance of the DLIPS method over the traditional fluorescence method using partial least squares (PLS) analysis. Additional multivariate analyses are explored, and the relevant photochemistry is elucidated in the context of perturbation wavelengths. We conclude that DLIPS is a promising biosensing approach with potential for in vivo analysis given the current findings with fluorophores relevant to biological tissues.     

Boron-containing two-photon-absorbing chromophores. 3. One- and two-photon photophysical properties of p-carborane-containing fluorescent bioprobes

Boron-containing two-photon-absorbing fluorophores have been prepared as new bifunctional molecules, potentially useful in two-photon excited microscopy (TPEM) and boron neutron capture therapy. They are based on a one-dimensional conjugated system containing a p-carborane entity at one end of the molecule and various electron-donating groups containing oxygen or nitrogen atoms at the other end. We investigated their one- and two-photon photophysical properties. They showed efficient fluorescence in an organic solvent, as well as in water for two of them, allowing microscopy on cell cultures. High two-photon absorption cross sections were determined in the 700-900 nm range. TPEM images were obtained with these new p-carborane-containing fluorophores, with laser intensities in the submilliwatt range.     

Amino(oligo)thiophene-based environmentally sensitive biomembrane chromophores

There is a growing need for cellular imaging with fluorescent probes that emit at longer wavelengths to minimize the effects of absorption, autofluorescence, and scattering from biological tissue. In this paper a series of new environmentally sensitive hemicyanine dyes featuring amino(oligo)thiophene donors have been synthesized via aldol condensation between a 4-methylpyridinium salt and various amino(oligo)thiophene carboxaldehydes, which were, in turn, obtained from amination of bromo(oligo)thiophene carboxaldehyde. Side chains on these fluorophores impart a strong affinity for biological membranes. Compared with benzene analogues, these thiophene fluorophores show significant red shift in the absorption and emission spectra, offering compact red and near-infrared emitting fluorophores. More importantly, both the fluorescence quantum yields and the emission peaks are very sensitive to various environmental factors such as solvent polarity or viscosity, membrane potential, and membrane composition. These chromophores also exhibit strong nonlinear optical properties, including two-photon fluorescence and second harmonic generation, which are themselves environmentally sensitive. The combination of long wavelength fluorescence and nonlinear optical properties make these chromophores very suitable for applications that require sensing or imaging deep inside tissues.     

Push-pull amino succinimidyl ester thiophene-based fluorescent dyes: synthesis and optical characterization

The design and synthesis of new fluorescent dyes with emission range at 490-650 nm are described. Their structural and electronic properties have been characterized by both experimental techniques and quantum-chemical calculations. The chromophores are donor-π-bridge-acceptor push-pull compounds with a π bridge of phenyl and thiophene rings and their combination. Compared with previous thiophene fluorophores, these dyes show significant redshift in the absorption and emission spectra and offer compact, red-emitting fluorophores. The dyes have amino succinimidyl active ester and can be readily conjugated to proteins, polymers and other amino-group-containing materials.     

Photophysics and nonlinear absorption of peripheral-substituted zinc phthalocyanines

The photophysical properties, such as the UV-vis absorption spectra, triplet transient difference absorption spectra, triplet excited-state extinction coefficients, quantum yields of the triplet excited state, and lifetimes of the triplet excited state, of 10 novel zinc phthalocyanine derivatives with mono- or tetraperipheral substituents have been systematically investigated in DMSO solution. All these complexes exhibit a wide optical window in the visible spectral range and display long triplet excited-state lifetimes (140-240 mus). It has been found that the complexes with tetrasubstituents at the alpha-positions exhibit a bathochromic shift in their UV-vis absorption spectra, fluorescence spectra, and triplet transient difference absorption spectra and have larger triplet excited-state absorption coefficients. The nonlinear absorption of these complexes has been investigated using the Z-scan technique. It is revealed that all complexes exhibit a strong reverse saturable absorption at 532 nm for nanosecond and picosecond laser pulses. The excited-state absorption cross sections were determined through a theoretical fitting of the experimental data using a five-band model. The complexes with tetrasubstituents at the alpha-positions exhibit larger ratios of triplet excited-state absorption to ground-state absorption cross sections (sigma T/sigma g) than the other complexes. In addition, the wavelength-dependent nonlinear absorption of these complexes was studied in the range of 470-550 nm with picosecond laser pulses. All complexes exhibit reverse saturable absorption in a broad visible spectral range for picosecond laser pulses. Finally, the nonlinear transmission behavior of these complexes for nanosecond laser pulses was demonstrated at 532 nm. All complexes, and especially the four alpha-tetrasubstituted complexes, exhibit stronger reverse saturable absorption than unsubstituted zinc phthalocyanines due to the larger ratio of their excited-state absorption cross sections to their respective ground-state absorption cross sections.     

Spectral characterization of chlorophyll fluorescence in barley leaves during linear heating. Analysis of high-temperature fluorescence rise around 60 degrees C

The spectral characteristics of chlorophyll fluorescence and absorption during linear heating of barley leaves within the range 25-75 degreesC (fluorescence temperature curve, FTC) were studied. Leaves with various content of light harvesting complexes (green, Chl b-less chlorina f2 and intermittent light grown) revealing different types of FTC were used. Differential absorption, emission and excitation spectra documented four characteristic phases of the FTC. The initial two FTC phases (a rise in the 46-49 degreesC region and a subsequent decrease to about 55 degreesC) mostly reflected changes in the fluorescence quantum yield peaking at about 685 nm. A steep second fluorescence rise at 55-61 degreesC was found to originate from a short-wavelength Chl a spectral form (emission maximum at 675 nm) causing a gradual blue shift of the emission spectra. In this temperature range, a clear correspondence of the blue shift in the emission and absorption spectra was found. We suggest that the second fluorescence rise in FTC reflects a weakening of the Chl a-protein interaction in the thylakoid membrane.     

Detection of Bacillus endospores using total luminescence spectroscopy

Detection and analysis of bacteria from environmental samples (e.g. water, air, and food) are usually accomplished by standard culture techniques or by analyses that target specific DNA sequences, antigens or chemicals. For large cell numbers in aqueous suspensions, an alternative technique that has proven useful is total luminescence spectroscopy (TLS). TLS is the acquisition of fluorescence data that records the unique excitation-emission matrix (EEM) of compound fluorophores. Past work has shown that one type of bacterial endospore, Bacillus megaterium, possessed a distinct EEM pattern useful for differentiating it in complex biological fluids and suspensions. The work described here extends those observations to establish some limits on the sensitivity and specificity of TLS for the detection and analysis of bacterial endospores versus (bacterial) vegetative cells in aqueous culture. Our findings show Bacillus endospores exhibit a dramatic blue shift of 130 nm in excitation and a smaller shift of 50 nm in emission when compared to ancillary endospore and non-endospore forming bacterial cells.