Thiol-Capped CdTe Quantum Dots with Two-Photon Excitation for Imaging High Autofluorescence Background Living Cells

To effectively image living cells with quantum dots (QDs), particularly for those cells containing high content of native fluorophores, the two-photon excitation (TPE) with a femto-second 800 nm laser was employed and compared with the single-photon excitations (SPE) of 405 nm and 488 nm in BY-2 Tobacco (BY-2-T) and human hepatocellular carcinoma (QGY) cells, respectively. The 405 nm SPE produced the bright photoluminescence (PL) signals of cellular QDs but also induced a strong autofluorescence(AF) from the native fluorophores like flavins in cells. The AF occupied about 30% and 13% of the total signals detected in QD imaging channel in the BY-2-T and QGY cells, respectively. With the excitation of 488 nm SPE, the PL signals were lower than those excited with the 405 nm SPE, although the AF signals were also reduced. The 800 nm TPE generated the best PL images of intracellular QDs with the highest signal ratio of PL to AF, because the two-photon absorption cross section of QDs is much higher than that of the native fluorophores. By means of the TPE, the reliable cellular imaging with QDs, even for the cells having the high AF background, can be achieved.     

Correlation Between Autofluorescence Intensity and Tumor Area in Mice Bearing Renal Cell Carcinoma

Protoporphyrin IX (PpIX) is a porphyrin derivative that is accumulated in cancerous tissue in consequence of the tumor-specific metabolic alterations. The aim of this study was to evaluate the accumulation of PpIX in mice bearing renal cell carcinoma by spectroscopy analysis. A total of 24 male Balb/c mice, 6 weeks old, were divided into six groups: Normal (without inoculation of tumor cells) and 4, 8, 13, 16, and 20 days after inoculation of tumor cells. The orthotopic tumor model of renal cancer was used. Murine renal cell carcinoma (Renca cells) were inoculated into the subcapsular space of the kidney. Normal and tumor-bearing kidneys in different progression stages were removed and analyzed by ex-vivo spectroscopy and by microscopy, for tumor histometric analysis. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences between normal and tumor-bearing kidneys in autofluorescence shape occurred in the 600–700 nm spectral region. A good correlation was found between emission band intensity at 635 nm and the tumor area.     

Sensing cell metabolism by time-resolved autofluorescence

We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular autofluorescence. The experiments on monolayered cell cultures show that with UV excitation at 365 nm the time-resolved autofluorescence decays, dominated by free-bound reduced nicotinamide adenine dinucleotide signals, are sensitive indicators for cell metabolism. However, the sensitivity decreases with the increase of excitation wavelength possibly due to the interference from free-bound flavin adenine dinucleotide fluorescence. The results demonstrate that time-resolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial precancer.     

High Sensitivity,Quantitative Measurements of Polyphosphate Using a New DAPI-Based Approach

Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4′,6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the μg/ml range. Here, we report that long-wavelength excitation (≥400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.     

Erythrocyte Protoporphyrin Fluorescence as a Biomarker for Monitoring Antiangiogenic Cancer Therapy

Renal cell carcinoma (RCC) remains one of the greatest challenges of urological oncology and is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and are amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of erythrocyte PpIX fluorescence spectroscopy for monitoring the efficacy of antiangiogenic therapy in metastatic renal cell carcinoma (mRCC), using an orthotopic metastatic mouse model. Balb/C-bearing Renca cells were treated with NIH/3T3-LendSN cells. Lung weight, nodule area, microvascular area (MVA), and erythrocyte PpIX fluorescence were evaluated. Emission spectra were obtained by exciting the samples at 405 nm. There was a significant decrease in lung wet weight, lung nodule area and MVA in the treated group compared to the control group (P < 0.001). Significant differences in autofluorescence shape were observed in the 620–650 nm spectral region. The most intense fluorescence peak was observed at ∼632 nm. The autofluorescence of the control samples was about 53% higher than that of normal blood (P < 0.05). In the group treated with ES, the autofluorescence was about 54% lower than in the control group (P < 0.05). Fluorescence intensity was positively correlated with the nodule area (R ² = 0.8859; P < 0.001) and MVA (R ² = 0.9431; P < 0.001) in the ES-treated group. These results demonstrate that the spectroscopic analysis method allows a selective detection of tumor masses. This preliminary study suggests that PpIX fluorescence may be useful as a biomarker for antiangiogenic cancer therapy.     

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.     

Study of Blood Porphyrin Spectral Profile for Diagnosis of Tumor Progression

Renal cell carcinoma (RCC) accounts for approximately 3% of new cancer incidence and mortality in the United States. Unfortunately many RCC masses remain asymptomatic and nonpalpable until they are advanced. Diagnosis and localization of early carcinoma play an important role in the prevention and curative treatment of RCC. The autofluorescence of blood porphyrin of healthy and tumor induced in male SCID mice was analyzed using fluorescence and excitation spectroscopy. A significant contrast between normal and tumor blood could be established. Blood porphyrin fluorophore showed enhanced fluorescence band (around 630 nm) in function of the tumor growth. This indicates that either the autofluorescence intensity of the blood fluorescence may provide a good parameter for the “first approximation” characterization of the tumor stage.     

Nonlinear Excitation of Tryptophan Emission Enhanced by Silver Nanoparticles

We measured and analyzed the behavior of the fluorescence of tryptophan water solutions with and without silver nanoparticles, excited by one, two and three photon processes. Two different colloids with silver nanoparticles with distinct diameters (0.65 nm and 9 nm) were used in the experiments. Fluorescence quenching was observed with one and two photon excitation. However, upon three-photon excitation, significant fluorescence enhancement was observed in the colloid. In this case excitation of the amino acid is assisted by the nonlinear absorption of infrared light by the silver nanoparticles. In this paper we are proposing a new way to explore metallic nanoparticles to enhance autofluorescence of biomolecules.     

Shifting of Fluorescence Peak in CdS Nanoparticles by Excitation Wavelength Change

CdS nanoparticles with different size are prepared by chemical bath deposition method. These particles show strong fluorescence at emission wavelength of 507 nm. It has been observed that this emission peak changes through a range of 147 nm, by varying the excitation wavelengths through 370–480 nm.The emission peak can thus be tuned by varying the excitation wavelengths. This peak emission wavelength shift is due to the selective excitation of vibronic levels in the surface state of the CdS nanoparticles.     

肝、肝癌及肝纤维细胞的荧光光谱及其荧光饱和强度分析

对肝细胞及肝病变细胞进行荧光光谱特性研究,能为早期筛查与攻克肝癌提供光谱学依据。实验目的包括,通过光谱实验获得细胞特异性荧光光谱;结合流式细胞仪获得最大饱和荧光强度与细胞直径的相关性。实验过程,首先使用荧光光谱仪来检测肝细胞、肝纤维细胞以及两种肝癌细胞;采用去拉曼散射的方法消除背景噪声,获得五种浓度下细胞荧光光谱;其次,使用流式细胞仪检测四种细胞直径的大小,根据双参数散点图分析四种细胞的直径特点;最后,利用高斯多峰拟合对比光谱差异,并且分析细胞直径对荧光饱和强度变化趋势的影响,得出细胞大小对荧光饱和强度非线性拟合曲线的影响规律。光谱实验发现,在550~750 nm之间,肝细胞存在两个特异性荧光峰,第一个峰值位于592 nm处,第二个峰位于682 nm处,且前者明显高于后者;肝癌,肝纤维细胞除具备与肝细胞相同位置的两个峰外,在726 nm处出现第三个特异性荧光峰,并在592 nm处获得最大激发光强,在726 nm处的荧光峰高于在682 nm处的第二个荧光峰;结合高斯多峰拟合法对峰值和各峰位置,以及峰型展宽进行分析。肝癌细胞和肝纤维细胞三个峰的展宽基本相同,正常肝细胞最大激发峰展宽略小于另三种细胞,但是682 nm处的小峰展宽略大于病变细胞;流式细胞仪实验结果显示,肝癌细胞直径大于肝纤维细胞大于肝细胞,同种浓度下荧光强度也是肝癌细胞高于肝纤维细胞高于肝细胞;利用非线性曲线拟合细胞最大荧光强度随浓度变化曲线,根据曲线斜率的变化规律,发现四种细胞的最大荧光强度会随着细胞浓度增大而增强,但是逐渐呈现荧光饱和状态。随着细胞直径增加,最大荧光强度饱和趋势更为明显,单个细胞自激发效率降低。结果显示,将细胞形态学与光谱学有机的融合,结合两种分析方式,能提高细胞判断的准确性和有效性。通过对肝细胞、肝癌细胞以及肝纤维细胞的荧光光谱特性进行研究,并结合细胞直径分析荧光饱和变化趋势,能够为肝病变细胞的研究提供一定的光谱学依据。     

Luminescent silicon nanoparticles with magnetic properties – production and investigation

Silicon nanoparticles (nSi) with unusual properties were studied. After suggested treatment they became luminescent and also acquired a magnetic moment. Nanoparticles were prepared by laser pyrolysis of silane in a gas flow reactor followed by chemical treatment in methanol (MeOH) + HF + FeCl₃ solution. After the treatment. nanoparticles gained stable luminescence with the peak position dependence on the excitation wavelength. With increasing of the excitation wavelength from 365 to 456 nm, the photoluminescent peak shifted from 632 to 665 nm. Luminescence of such nanoparticles had blue shift in comparison with the nanoparticles etched in widely-used solution for the silicon—MeOH + HF + HNO₃. Moreover, after such treatment the magnetic moment of nanoparticles appeared, which is not inherent for the as-prepared nSi. Multifunctional silicon nanoparticles with both stable luminescence and magnetic moment at the same time are perspective for biology and medicine use as the optical and magnetic markers.     

Multi-wavelength generation using a bismuth-based EDF and Brillouin effect in a linear cavity configuration

The generation of a multiple wavelength source using a bismuth-based erbium-doped fiber (Bi-EDF) in a linear cavity configuration is demonstrated. The configuration uses a pair of optical circulators at the input and output ends of the cavity to form a resonator for multi-wavelength generation in conjunction with optical couplers to inject the Brillouin pump (BP) and to tap the output at the two ends. The Brillouin erbium fiber laser (BEFL) comb with a 2.15 m of Bi-EDF has a wavelength spacing of 0.09 nm and operates in long-wavelength (L-) band region. A stable output laser comb of 50 lines is obtained at a BP of 1568.2 nm and 5 dBm and two 1480 nm pumps at 120 mW. The injected BP wavelength and power as well as the 1480 nm pump powers have a great effect on the number of lines and output power of the BEFL. This configuration is compact due to the use of the significantly shorter Bi-EDF as the linear gain medium, and can be made more compact by replacing the single-mode fiber with highly non-linear fibers such as holey fibers.     

Naturally grown Ag nanoparticles on quartz substrates as SERS substrate excited by a 488 nm diode laser system for SERDS

A silver nanoparticle ensemble was prepared under ultrahigh vacuum (UHV) conditions by Volmer–Weber growth on a quartz substrate for surface-enhanced Raman scattering (SERS) investigations of pyrene molecules. To tune the surface plasmon resonance frequency in the vicinity of the excitation wavelength of 488 nm of the diode laser, the morphology of the silver nanoparticles was optimized. The substrates were mounted in a flow-through cell as part of the optical Raman set-up. A microsystem diode laser generates two slightly different emission wavelengths (λ=487.61 nm and λ=487.91 nm) with a spectral width <10 pm and an optical power of 20 mW, i.e. SERS experiments are possible but also shifted excitation Raman difference spectroscopy (SERDS) can be carried out. For trace analysis of pyrene in water we demonstrate SERS/SERDS experiments which lead to a limit of detection of 2 nmol/l for pyrene. These results suggest that with silver nanoparticle ensembles excited at their plasmon resonance at 488 nm combined SERS/SERDS measurements can be effectively performed for in-situ trace analysis of pollutant chemicals in water.     

Spectrofluorimetric Methods for the Determination of Gemifloxacin in Tablets and Spiked Plasma Samples

Two new, sensitive and selective spectrofluorimetric methods have been developed for the determination of gemifloxacin (GFX) in tablets and spiked plasma samples. Gemifloxacin, as a primary amine compound, reacts with 7-chloro-4-nitrobenzofurazon (NBD-Cl) (for method A) and fluorescamine (for method B) which are a highly sensitive fluorogenic reagents used in many investigations. For method A, the reaction product was measured spectrofluorimetrically at 516 nm with excitation at 451 nm. The reaction proceeded quantitatively at pH 8.5, 80 °C in 7 min. For method B, the method was based on the reaction between GFX and fluorescamine in borate buffer solution of pH 8.5 to give highly fluorescent derivatives that were measured at 481 nm using an excitation wavelength of 351 nm. The fluorescence intensity was directly proportional to the concentration over the range 40–200 ng mL⁻¹ and 100–1,200 ng mL⁻¹ for method A and B, respectively. Successful applications of the developed methods, for the drug determination in pharmaceutical preparations and spiked plasma samples, were performed.     

Influence of argon ambience on the structural,morphological and optical properties of pulsed laser ablated zinc sulfide thin films

Nanostructured zinc suplhide thin films are successfully deposited on quartz substrates using pulsed laser deposition (PLD) under different argon pressures (0, 5, 10, 15 and 20 Pa). The influence of argon ambience on the microstructural, optical and luminescence properties of zinc sulfide (ZnS) thin films is systematically investigated. The GIXRD data suggests rhombohedral structure for ZnS films prepared under different argon ambience. Self-assembly of grains into well-defined patterns along the y direction is observed in the AFM image of the film deposited under argon pressure 20 Pa. All the films show a blue shift in optical band gap. This can be due to the quantum confinement effect and less widening of conduction and valence band for the films with less thickness and smaller grain size. The PL spectra of the different films are recorded at excitation wavelengths 250 nm and 325 nm and the spectra are interpreted. The PL spectra of the films recorded at excitation wavelength 325 nm show intense yellow emission. The film deposited under an argon pressure of 15 Pa shows the highest PL intensity for excitation wavelength 325 nm. For the PL spectra (excitation at 250 nm), the highest PL intensity is observed for the film prepared under argon free ambience. In our study, 15 Pa is the optimum argon pressure for better crystallinity and intense yellow emission when excited at 325 nm.     

基于激光共焦扫描术的亚心形扁藻显微图像与光谱

采用激光共聚焦扫描显微技术,针对亚心形扁藻开展了研究.从获得的488 nm Ar+激光单光子激发的亚心形扁藻自体荧光光谱与图像,可知细胞内有一杯状叶绿体物质,其荧光峰值为682 nm,对应叶绿体发出的红色荧光.在单通道模式下,获得800 nm fs激光双光子激发的扁藻自体荧光光谱与图像,可知每个杯状叶绿体的内部有一个自体荧光更强的圆形物质.在双通道模式下,可分别获得小圆形物质的自体荧光图像,杯状叶绿体自体荧光图像,以及两个通道图像的叠加.进一步获得了双光子藻细胞荧光图的6个主要的荧光峰.采用单光子激光激发可获得亚心形扁藻叶绿体自体荧光图像及其荧光光谱,而双光子激光激发荧光光谱的多通道以及Lambda模式下采集光谱信号与图像,不仅可观察到亚心形扁藻的内部形态结构,还可能从双光子激发荧光图中研究分析亚心形扁藻生化物质的存在,灵敏度较单光子激发高.激光扫描共聚焦显微技术,特别是双光子荧光与图像技术可为海藻的检测与研究提供一种快速、实时、有效、简便的方法.     

Photoluminescence characterization of beryllium-implanted 6H–silicon carbide

Beryllium has been implanted into both n- and p-type 6H–SiC with post-implantation annealing at 1600 °C. Photoluminescence (PL) measurements have been performed, and PL lines at 420.5, 431 nm, and a broad band at around 505 nm have been observed. The line at 420.5 nm is attributed to an intrinsic defect D_II-center induced by beryllium implantation. The effects of excitation intensity and temperature during the PL experiments are investigated. Based on the excitation laser dependence PL result, the new doublet lines at around 431 nm are thought to be associated with beryllium related bound excitons. The broad band corresponding to the green luminescence at room temperature has been attributed to the recombination of free carriers to beryllium bound levels.     

Two-Color Two-Photon Fluorescence Laser Scanning Microscopy

We present the first realization of a Two-Color Two-Photon Laser-Scanning Microscope (2c2pLSM) and UV fluorescence images of cells acquired with this technique. Fluorescence is induced by two-color two-photon absorption using the fundamental and the second harmonic of a Ti:Sa femtosecond laser. Simultaneous absorption of an 800 nm photon and a 400 nm photon energetically corresponds to one-photon absorption at 266 nm. This technique for Laser-Scanning Microscopy extends the excitation wavelength range of a Ti:Sa powered fluorescence microscope to the UV. In addition to the known advantages of multi-photon microscopy like intrinsic 3D resolution, reduced photo damage and high penetration depth 2c2pLSM offers the possibility of using standard high numeric aperture objectives for UV fluorescence imaging. The effective excitation wavelength of 266 nm corresponds especially well to the excitation spectrum of tryptophan. Hence, it is an ideal tool for label free fluorescence studies and imaging of intrinsic protein fluorescence which originates mainly from tryptophan. Thus a very sensitive natural lifetime probe can be used for monitoring protein reactions or changes in conformation. First measurements of living MIN-6 cells reveal differences between the UV fluorescence lifetimes of the nucleus and cytoplasm. The significance of this method was further demonstrated by monitoring the binding of biotin to avidin.     

Fluorescence Spectroscopy for Diagnostic Differentiation in Uteri’s Cervix Biopsies with Cervical/Vaginal Atypical Cytology

This work aims the diagnostic differentiation of chronic inflammation (CC), low-grade Intraepithelial squamous lesions (LGSIL) and high-grade intraepithelial squamous lesions (HGSIL) in biopsies of cervix of uterus from patients with atypias (ASC-US and ASC-H) and lesions (LGSIL and HGSIL), traced in the cervical/vaginal cytology by using Laser-Induced Fluorescence Spectroscopy (LIFS), with 488 nm excitation wavelength. Ninety seven biopsies from 32 patients with atypical cervical/vaginal cytology were collected. The biopsies were guided by colposcopy and taken at the squamous-columnar junction. Fluorescence emission spectra of each biopsy were collected by means of an optical fiber cable coupled to an argon laser at 488 nm as excitation source and addressed to a spectrograph and CCD camera/controller. Spectra were separated into three groups, CC, LGSIL and HGSIL, based on the cytopathology. It was detected similar mean spectra profiles for CC and LGSIL, and differences for HGSIL. An algorithm was developed for tissue classification based on the intensity of the multiplication of each spectrum by the mean spectrum of each group, searching for a discriminator that would address this spectral difference. The sensitivity and specificity of HGSIL identification, compared to CC and LGSIL was 89% and 100%, respectively. The LIFS using excitation wavelength of 488 nm could be used to differentiate HGSIL lesions from LGSIL and CC inflammation, and could help a precocious and less invasive diagnosis of cervix lesions.     

Optical limiting characteristics of Dichloridobis(1-ethyl-2, 6-dimethylpyridinium-4-olate-κO) zinc (II)

A newly synthesized metal-organic co-ordination compound Dichloridobis (1-ethyl-2,6-dimethylpyridinium-4-olate-κO) zinc(II) (EDMPZC) is characterized by FTIR to confirm the molecular structure. It crystallizes in the monoclinic system with the centrosymmetric space group C2/c and the unit cell parameters are determined from the single crystal X-ray diffraction analysis. Optical energy band gap is found to be 3.5 eV by UV-Visible absorption studies. The third order nonlinear optical properties were investigated by open-aperture Z-scan measurements using Nd:YAG laser (532 nm, 5 ns). Two-photon absorption and reverse saturable absorption mechanisms at the excitation wavelength are found to cause the observed optical limiting behavior.