Surface-sensitive polarized Raman spectroscopy of biological tissue

In a two-layer diffusing medium, polarized light directly backscattering off the superficial layer will partially retain its sense of polarization, whereas deeper-probing light will be increasingly depolarized by diffusion. This effect has been studied in both elastic scattering and fluorescence contexts. We apply this method to Raman scattering in two two-layer models with a highly diffusing lower layer of glucose powder and an upper layer of either clear plastic or chicken skin. We employ detection of orthogonal polarization states to generate a Raman spectrum of only the superficial layer by combining the orthogonal signals.     

Investigation of transient two-photon excited fluorescence in biological tissue

The fluorescence power from biological tissue excited by a femtosecond laser pulse compared with excitation power does not appear to obey a simple quadratic relationship given by the steady non-linear theory.A more reliable analysis is developed based on transient two-photon absorption because the response time of two-photon absorption is longer than the width of a femtosecond pulse.Good agreement is obtained between the theoretical analysis and the experimental results of fluorescence power versus excitation power.This letter offers potential value to non-linear optics in biological tissues.     

Simultaneous time- and wavelength-resolved fluorescence spectroscopy for near real-time tissue diagnosis

A novel fiber-optic-based method for simultaneous time- and wavelength-resolved fluorescence spectroscopy for the rapid diagnosis of diseased tissue is demonstrated. By combining multiple bandpass and dichroic filters (405/40, 460/50, and 550/50) with different lengths of optical fiber (1, 10, and 19 m) acting as an optical delay this system enables the near real-time acquisition and characterization of time-resolved fluorescence spectra using a single detector and excitation input. The recording of multiple fluorescence response pulses at selected wavelengths can be completed in hundreds of nanoseconds, which provides the capability of a real-time characterization of biological systems.     

Excimer laser spectroscopy: influence of tissue ablation on vessel wall fluorescence

Limited steerability and injury to the normal vessel wall are major drawbacks of laser coronary angioplasty. To overcome these limitations a new generation of laser systems has been developed which allows not only to eliminate the atherosclerotic plaque but to guide the laser beam by analyzing the laser induced tissue fluorescence (= spectroscopy) for the treatment of the atherosclerotic vessel. An excimer laser (MAX 10 LP, 308 nm, Technolas, Munich, Germany) was used with an emitting (phi 1070 microns) and a detecting (phi 130 microns) optical fiber to induce tissue fluorescence which was analyzed quantitatively by a computerized system. Specimens from the descending (thoracic) aorta were obtained from 24 patients (mean age 68.1 years, range 44-92). Tissue fluorescence was induced with ablating (26-30 mJ/mm2) and nonablating (3 mJ/cm2) laser activations. The emitted fluorescence (range 380-575 nm) was normalized to a wavelength of 380 nm; as a measure of tissue fluorescence the intensity ratio at 500 nm divided by 400 nm was calculated in normal (n = 78), mildly atherosclerotic (n = 40), and severely atherosclerotic (n = 48) tissue samples. Repeated laser activations were carried out and tissue fluorescence was checked until the fluorescence spectrum was normalized. All tissue samples were analyzed histologically by a semiquantitative score. Normal tissue samples showed the highest intensity ratios (5.9 +/- 3.4), whereas mildly (2.9 +/- 1.3) and severely atherosclerotic (2.1 +/- 1.0) samples elicited a significantly reduced fluorescence. Repeated tissue ablations were associated with a normalization of fluorescence intensity ratios in the mildly (7.0) as well as in the severely diseased (4.9) vessels. A curvilinear relationship between intensity ratio and the semiquantitative score was observed (r = 0.66) as well as between intensity ratio and intimal wall thickness (r = 0.62). No gender related differences were found but there was an inverse relationship between fluorescence intensity ratio and age (r = 0.56) as well as between intimal thickness and age (r = 0.41). Excimer laser spectroscopy allows reliable detection of atherosclerotic vessel alterations. Fluorescence intensity ratio is inversely proportional to the intimal wall thickness and the severity of the histologic alterations. There is an age dependency of fluorescence intensity ratio which can be explained by an increase in intimal wall thickness. Successful tissue ablation can be obtained by laser angioplasty and allows determination of the optimal point where complete tissue ablation is achieved by laser activation. Thus, excimer laser spectroscopy is an effective method for selective tissue ablation by laser angioplasty.     

双光子激发时间分辨荧光光谱测量技术

建立了一台基于高重复频率扫描相机的双光子激发时间分辨荧光光谱测量系统，能够同时测量样品的荧光光谱和寿命. 该系统的时间分辨率为6.5—200ps，光谱分辨率为1—3nm，能够实现快速数据采集以及可靠和可重复的寿命和光谱测量. 利用标准荧光染料(若丹明6G、香豆素314)及其混合溶液对该系统进行了测试，所得到的荧光光谱分布和寿命值与文献报道一致. 实验结果表明，该系统能有效区分多组分荧光团. 这为鉴别多荧光团或多组分生物组织提供了一种独特的对比方法，可用于多光谱分辨荧光寿命成像和荧光共振能量转移成像等方面. 关键词： 荧光寿命 荧光光谱 双光子激发 高重复频率扫描相机     

Two-photon standing-wave fluorescence correlation spectroscopy

A fluorescence correlation spectroscopy experiment that combines two-photon excitation and a standing-wave interference pattern is presented. The experimental correlation function can be analyzed using a simple expression involving (1) an exponential decay with time constant tau(f), which reflects diffusion across the interference fringes, and (2) a longer-lived decay with time constant tau(omega), which reflects diffusion in and out of the focal spot. The diffusion of Rhodamine 110 in water and ethylene glycol is measured using this method. The ability to simultaneously measure diffusion on two different time and lengthscales makes this experiment especially useful in environments where anomalous diffusion is suspected.     

Laser induced fluorescence spectroscopy of NdO

Laser induced fluorescence spectra of ¹⁴²NdO have been excited using both fixed frequency argon ion and tunable ring dye lasers and detected at high resolution with a Fourier transform spectrometer. Nine low lying electronic states resulting from the Nd²⁺(4f³6s)O²⁻ configuration were detected of which four, the second lowest , 3, and 5 states, (2)2, (2)3, (2)5, and the lowest state, (1)6, have been observed for the first time. In addition, new vibrational levels were observed in the lowest , (1)5 (v=1) and second lowest , (2)4 (v=1, 2) states. Abnormally large Ω doubling in both states has been attributed to interactions involving neighboring and 0 states. Several perturbations were observed and used as an aid in assigning some of the states. Both the order and energies of the low lying states have been shown to be consistent with Ligand Field theory calculations. Rotational relaxation in several of the spectra has allowed calculation of accurate rotational constants for several states while, for other states, approximate parameters have been calculated from combination differences.     

Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy

This didactic paper summarizes the mathematical expressions needed for analysis of fluorescence anisotropy decays from polarized frequency-domain fluorescence data. The observed values are the phase angle difference between the polarized components of the emission and the modulated anisotropy, which is the ratio of the polarized and amplitude-modulated components of the emission. This procedure requires a separate measurement of the intensity decay of the total emission. The expressions are suitable for any number of exponential components in both the intensity decay and the anisotropy decay. The formalism is generalized for global analysis of anisotropy decays measured at different excitation wavelengths and for different intensity decay times as the result of quenching. Additionally, we describe the expressions required for associated anisotropy decays, that is, anisotropy decays where each correlation time is associated with a decay time present in the anisotropy decay. And finally, we present expressions appropriate for distributions of correlation times. This article should serve as a reference for researchers using frequency-domain fluorometry.     

Polarized fluorescence spectroscopy of human tissues

We report the results of a study carried out to investigate the effect of blood absorption on polarized and unpolarized fluorescence from resected tissue samples and tissue phantoms. The signatures of blood absorption were found to be significantly smaller in polarized fluorescence than in unpolarized fluorescence spectra. The reduced effect of blood absorption on polarized fluorescence also leads to reduced site-to-site variability in polarized fluorescence intensity and line shape compared with unpolarized fluorescence.     

Terahertz spectroscopy of biological molecules

We study experimentally the absorption and refraction spectra for a number of amino acids, proteins and nucleic acids in the frequency range 0.1–3.5 THz. The characteristic absorption lines are revealed. The spectroscopy of water solutions of proteins and nucleic acids is carried out under conditions of total internal refraction. This method can be used for studies of the functional properties of biomolecules and the dynamics of their interaction.     

Study on temperature & EMF co-effects on insulin conformation and biological functions by fluorescence and Raman spectroscopy

Our previous studies had suggested that the intercellular signal molecule might be an important target of electromagnetic fields. Insulin, an intercellule signal molecule, plays a critical role in transferring life information. The studies on effects of pulsed electric fields (PEF) on insulin molecule are meaningful for explaining the mechanism of biological effects of electromagnetic fields. The PEF, which we used, with its highest electric field (2 x 10(6) V x m(-1)) coupled into the insulin buffer, was about 1 V x cm(-1) cm, with a repeating frequency of 50 Hz. In the present study, the changes of insulin conformation induced by PEF were studied by fluorescence spectroscopy. Insulin solution was exposed to 50 Hz PEF with different electric field intensities for 5-35 min, which caused a time-and dose-dependent decrease in fluorescence intensities of insulin. Further, insulin solution was exposed to PEF at different temperatures to investigate the effects of PEF co-operated with temperature on insulin. The results indicated that the difference in temperature (about 5 degrees C) could induce conflict results, which is due to the effects of PEF co-operated with temperature rather than only to the effect of temperature. The authors calculated that the increase in temperature induced by PEF was 0.07 degrees C (less than 0.1 degrees C). So the effects of PEF were scarcely explained by thermal effects, it belongs to "non-thermal effects" of electric fields. So it was concluded that temperature is a considerably important factor in "non-thermal effects" of electric fields, and the ignorance of variety of temperature probably result in the contrary conclusion. Further, Raman spectroscopy was used to investigate the details of structure of insulin treated by PEF co-operated with temperature. The results of Raman spectroscopy verified the effects of PEF co-operated with temperature on insulin. And the reductions of the S-S band intensity at 510 cm(-1), the skeletal C-C stretch band intensity at 934 cm(-1), and the content of the secondary structure of the alpha helix were observed. Both S--S linkages and alpha helix structure were important to the stabilization of insulin conformation. Modification of insulin may change the biological activity either by reducing the affinity of the hormone for the receptor or by decreasing the ability of the complex, when formed, to elicit a biological response.     

Triplet-state monitoring by fluorescence correlation spectroscopy

The effects of high excitation intensities in fluorescence correlation spectroscopy (FCS) in terms of saturation and triplet-state build-up have been studied for the case of Rh6G in aqueous solution. It was found that FCS provides a powerful means for the determination of intersystem crossing and triplet-state depopulation rates of fluorophores in solution.This is a peer-reviewed conference proceeding article from the Third Conference on Methods and Applications of Fluorescence Spectroscopy, Prague, Czech Republic, October 18–21, 1993.     

Laser-induced fluorescence spectroscopy of the secondary cataract

Excitation–emission matrices of laser-induced fluorescence of lens capsule epithelium, the lens nucleus, and the lens capsule are investigated. A solid-state laser in combination with an optical parametric generator tunable in the range from 210 to 350 nm was used for excitation of fluorescence. The spectra of fluorescence of all three types of tissues exhibit typical features that are specific to them and drastically differ from one another. This effect can be used for intrasurgical control of presence of residual lens capsule epithelium cells in the capsular bag after surgical treatment of a cataract.     

Photoelectron spectroscopy of some biological molecules

The ultraviolet photoelectron spectra (UPS) of the following biologically-active compounds are reported and/or assigned: 2,4-dinitrophenol, nicotinic acid, nicotinamide, barbituric acid, xanthine, hypoxanthine, uric acid, uracil, thymine, cytosine, adenine, guanine, β-carotene, menadione, purine and pyrimidine. The importance of UPS data is exemplified in two ways: First, by investigating the validity of the Pullman k-index approach ¹⁴ to ionization energies; and, second, by generating an experimental scale of electron-donating ability.     

Photophysical study on daunorubicin by fluorescence spectroscopy

Steady-state fluorescence and time-resolved fluorescence intensity decays of daunorubicin have been studied in polar solvents and in aqueous solution by a time-correlated single-photon counting technique. Daunorubicin, quinizarin, and 2,3-dimethylquinizarin show bi-exponential decay. The decay of daunorubicin becomes tri-exponential in the presence of adenosine 5′ monophosphate. The quenching of the fluorescence of daunorubicin by adenosine 5′ monophosphate exhibits downward deviation from the Stern-Volmer linearity, suggesting the existence of fluorophore in two conformers in the ground state differing only in the extent of hydrogen bonding.     

Confocal fluorescence spectroscopy and anisotropy imaging system

We report the design and implementation of a laser scanning confocal fluorescence system with spectroscopy and anisotropy imaging capabilities. Confocal spectroscopy is achieved with a fiber pinhole that is inserted into and removed from the detection path as needed. Fluorescence anisotropy imaging is accomplished with a polarizing beam splitter placed after the conventional pinhole. Two orthogonal polarizations are detected simultaneously with balanced photomultiplier tubes. The quality of the axial sectioning that is achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and examples of polarization-sensitive fluorescence imaging are demonstrated in tumor cell monolayers.     

Diagnosis of corneal pathology by laser fluorescence spectroscopy

We have studied the difference between the fluorescence spectra of the human cornea in vivo under normal conditions and after contact lenses have been worn for different lengths of time, with excitation by emission from a nitrogen laser (337 nm). The most significant sections of the difference spectrum were identified, corresponding to peaks for endogenous fluorophores (NADH and collagen). A high correlation was found between how long the contact lenses have been worn and the fluorescence intensity ratio for wavelengths 460 nm and 410 nm.