On the Nature of Stationary and Time-Resolved Fluorescence Spectroscopy of Collagen Powder from Bovine Achilles Tendon.

This paper presents a more systematic study of steady-state and time-resolved autofluorescence spectroscopy of collagen isolated from bovine Achilles tendon. In steady-state fluorescence measurements, the excitation and emission spectra of collagen powder, recorded at different fluorescence excitation and detection wavelengths, were compared with the fluorescence excitation and emission spectra of the amino acids phenylalanine, tyrosine, and tryptophan, as well as with similar spectra for 13 autofluorescent collagen cross-links, which have been identified and described in the literature so far. In time-resolved studies, fluorescence was excited by the pulsed light of different wavelengths, and for each excitation wavelength, fluorescence decay was recorded for several detection wavelengths. Data analysis allowed recovery of the fluorescence decay times for each experimental excitation detection event. The obtained information on the decay times of the measured fluorescent signals was discussed, taking into account the available literature data from similar studies of isolated collagen and collagen-rich tissues. Based on the obtained results, it was found that the shape and position of the measured fluorescence excitation and emission spectra of collagen strongly depend on the emission and excitation wavelengths selected in the measurements. From the recorded excitation and emission bands of collagen, it can be concluded with high probability that there are additional, so far unidentified, collagen cross-links, which can be excited at longer excitation wavelengths. In addition, the collagen excitation spectra were measured at longer emission wavelengths at which the collagen cross-links emit fluorescent light. In addition to the emission spectra obtained for excitation in the deep-UV region, the results of time-resolved fluorescence studies with excitation in the deep-UV region and detection at longer wavelengths suggest that fluorescence excitation energy transfer processes occur from the amino acids to the collagen cross-links, and also between the cross-links themselves.

Low-level laser irradiation modifies the effect of hyperglycemia on adhesion molecule levels.

Endothelium plays a key role in maintaining vascular homeostasis by secreting active factors involved in many biological processes such as hemostasis, angiogenesis, and inflammation. Hyperglycemia in diabetic patients causes dysfunction of endothelial cells. Soluble fractions of adhesion molecules like sE-selectin and vascular cell adhesion molecule (sVCAM) are considered as markers of endothelial damage. The low-level laser therapy (LLLT) effectively supports the conventional treatment of vascular complications in diabetes, for example hard-to-heal wounds in patients with diabetic foot syndrome. The aim of our study was to evaluate the effect of low-energy laser at the wavelength of 635 nm (visible light) and 830 nm (infrared) on the concentration of adhesion molecules: sE-selectin and sVCAM in the supernatant of endothelial cell culture of HUVEC line. Cells were cultured under high-glucose conditions of 30 mM/L. We have found an increase in sE-selectin and sVCAM levels in the supernatant of cells cultured under hyperglycemic conditions. This fact confirms detrimental influence of hyperglycemia on vascular endothelial cell cultures. LLLT can modulate the inflammation process. It leads to a decrease in sE-selectin and sVCAM concentration in the supernatant and an increase in the number of endothelial cells cultured under hyperglycemic conditions. The influence of LLLT is greater at the wavelength of 830 nm.

Autofluorescence spectroscopy in the differentiation of laryngeal epithelial lesions - preliminary results.

Conclusions Autofluorescence spectroscopy may be a supporting tool for differential diagnosis of changes in laryngeal epithelium. Objectives Early detection and differential diagnosis of proliferative changes in the larynx are still a challenge for laryngologists. The aim of the study was to evaluate the autofluorescence spectroscopy technique to in vitro differential diagnosis of pathological changes in the epithelium of the larynx. Methods Forty-two patients aged 34-79 years were included in the study. The fifty-two tissue specimens, including 10 samples of cancerous lesion, 10 adjacent normal tissue, 10 chronic inflammation, eight cyst, three leukoplakia, four polyp, and seven Reinke's edema, were obtained during laryngological procedures. All tissue samples were independently diagnosed histopathologically. The autofluorescence emission spectra at two excitation wavelengths, 290 nm and 370 nm, were measured for every sample studied. Results The autofluorescence signals of cancerous tissue samples at both excitations exhibited identical emission band shapes of much lower intensities at their maxima as compared to the adjacent healthy tissue samples studied. The autofluorescence spectra intensities of cancerous and normal tissues varied inter-individually. Evident differences in autofluorescence intensities and its band shapes of different pathological laryngeal changes at the 290 nm excitations were demonstrated.

Low-level laser irradiation effect on endothelial cells under conditions of hyperglycemia.

Diabetes mellitus is considered to be a very serious lifestyle disease leading to cardiovascular complications and impaired wound healing observed in the diabetic foot syndrome. Chronic hyperglycemia is the source of the endothelial activation. The inflammatory process in diabetes is associated with the secretion of inflammatory cytokines by endothelial cells, e.g., tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6). The method of phototherapy using laser beam of low power (LLLT-low-level laser therapy) effectively supports the conventional treatment of diabetic vascular complications such as diabetic foot syndrome. The aim of our study was to evaluate the effect of low-power laser irradiation at two wavelengths (635 and 830 nm) on the secretion of inflammatory factors (TNF-α and IL-6) by the endothelial cell culture-HUVEC line (human umbilical vein endothelial cell)-under conditions of hyperglycemia. It is considered that adverse effects of hyperglycemia on vascular endothelial cells may be corrected by the action of LLLT, especially with the wavelength of 830 nm. It leads to the reduction of TNF-α concentration in the supernatant and enhancement of cell proliferation. Endothelial cells play an important role in the pathogenesis of diabetes; however, a small number of studies evaluate an impact of LLLT on these cells under conditions of hyperglycemia. Further work on this subject is warranted.

Probing the use of fluorescence spectroscopy as a novel diagnostic tool in patients with rheumatoid arthritis: applicability in the detection of secondary amyloidosis.

BACKGROUND: Secondary amyloidosis is a frequently reported complication of rheumatoid arthritis. Currently, accepted diagnostic protocols for secondary amyloidosis involve histopathological and histochemical examinations of collected tissue specimens. The purpose of the current report was to evaluate the value of fluorescence spectroscopy as a supplementary tool in the diagnosis of secondary amyloidosis. MATERIAL/METHODS: Tissue specimens were collected from abdominal folds, gingiva or rectal mucosa of 99 patients affected with rheumatoid arthritis. Tissue samples were subjected to preliminary clinical observations, histopathological examinations and laboratory tests. These procedures were used to subdivide tissue samples into either amyloid-containing or amyloid-free control subgroups. All collected tissue samples were examined with the use of a designated spectrofluorometer and fluorescence spectral images were generated. RESULTS: It was found that fluorescence spectra for amyloid-containing tissues were typically characterized by a double emittance peak. In contrast, amyloid-free samples were characterized by fluorescence spectra with a single λmax value. Specimen collection site, age and sex did not appear to influence the morphology of electromagnetic spectra, which were generated for both amyloid-containing and amyloid-free tissue samples. The sensitivity of the fluorometric approach was ~78% and the specificity was 100%. Possible shortcomings of the technique may be due to the limit of detection of the instrument used. CONCLUSIONS: Fluorescence spectroscopy may potentially be used as an effective, instantaneous and low-cost diagnostic tool for suspected secondary amyloidosis in patients affected with rheumatoid arthritis.

Ultrastructural differences in rectus sheath of hernia patients and healthy controls.

BACKGROUND: The etiology of inguinal hernia remains unclear. Research data indicate the presence of pathologic alterations within the connective tissue; their exact character remains the subject of dispute. The search for new methods to diagnose connective tissue abnormalities, and thoroughly explain the character of the ultrastructural alterations, continues. MATERIALS AND METHODS: The study group included 10 male patients aged 18-60 y (five with primary inguinal hernia and five with acute appendicitis with no history of hernia). A specimen of the rectus muscle sheath was harvested from all of them upon surgery. The tissue samples were fixed and examined by spectrofluorometry and fluorescence microscopy, yielding fluorescence spectra and microscopic fluorescence images. RESULTS: Both techniques have demonstrated significant differences between the biopsy samples harvested from hernia patients and healthy controls. The groups of fluorescence spectra were shifted relative to each other and showed maximum emission at different wavelengths after excitation with 350 nm light (arbitrarily chosen for one of the cross-link proteins). The spectra obtained for healthy controls were more homogenous, while the spectra of the hernia samples differed even between each other. In microscopic images, the difference was a more chaotic distribution of fluorophores in the samples obtained from hernia patients. CONCLUSIONS: The evidence of significant differences between the samples harvested from the same location from hernia patients and healthy controls, found by fluorescence techniques, indicates the presence of abnormalities in the connective tissue forming the rectus muscle sheath. This area is not a part of the hernial defect, therefore, we can assume that the changes can be attributed to a generalized process.

Another treatment of fluorescence polarization microspectroscopy and imaging.

We here discuss a general (symmetry adapted) treatment for one-photon-excitation time-resolved fluorescence polarization microspectroscopy (TRFPM) at combined wide-angular excitation and detection apertures that correctly couples the principles of the optics of objective lenses with the principles of fluorescence spectroscopy with polarized light. The treatment is unified in the sense that it covers the electromagnetic description of focusing a linearly polarized beam of exciting light (diffraction theory, DT) and the description of the same problem in terms of the meridional plane properties (MPP) of the objective lenses (geometrical optics). It is shown that both approaches are quantitatively equivalent from the point of view of the polarization effects in typical TRFPM experiments on linear absorbers, despite the fact that in the MPP treatment the region of focus is treated as a pointlike object, while in the DT method the region of focus is characterized by a three-dimensional (3D) inhomogeneous electromagnetic field distribution, of generally ellipsoidal polarization at different points of the focus. This finding is essentially important from the point of view of the experimental practice because the MPP treatment is based on two very simple trigonometric expressions, in evident contrast to the DT method, in which the high-aperture focusing is described in terms of three complicated 3D integrals involving the Bessel functions of the first kind. A few words of comment are added on a similar problem in the case of nonlinear one-photon absorbers (e.g., chiral fluorophores). We discuss the synthetic fluorescence decays for the wide-field- and evanescent-wave-excitation confocal (or wide-field) detection fluorescence polarization microspectroscopy and imaging, which indicate the right experimental protocols for the kinetic and dynamic fluorescence polarization microspectroscopic studies. The manifestations of the effects resulting from the application of the wide-angular excitation and/or detection apertures are displayed and discussed in a systematic way. A few words of comment are added on the application of the symmetry adapted calibration (SAC) method to TRFPM experiments. A very important aim of this article is to provide a correct and more complete description of fluorescence polarization microspectroscopy and imaging of macroscopically isotropic media (i.e., solutions, solutions of labeled macromolecules, membrane suspensions, or biological cells), that can be immediately applied in the experimental practice in the life and medical sciences and also in different areas of nano(bio)technology.

Another look at magic-angle-detected fluorescence and emission anisotropy decays in fluorescence microscopy.

It is shown that in contrast to a traditional fluorescence spectroscopy with the parallel beams of light, in which the kinetic fluorescence decays are collected at the so-called magic-angle of thetamag=54.7 degrees, in the fluorescence microscopy, the value of the magic-angle depends on the numerical aperture (NA) of a microscope objective and on the refractive index (n) of an immersion liquid used. Two methods enabling the determination of the magic-angle values corresponding to different values of NA/n, are discussed. It is shown that thetamag changes from a value of 54.7 degrees at the NA/n-->0, to a value of 45 degrees with NA/n-->1. Also in contrast to a traditional fluorescence spectroscopy, in the fluorescence microscopy the term I parallel(t)+2I perpendicular (t) does not represent the total fluorescence intensity Itot(t), because the resulting fluorescence decay I parallel(t)+2I perpendicular (t) is contributed by the dynamic evolution of excited fluorophores. A correctly defined total fluorescence intensity solely represents the kinetic evolution of excited fluorophores, and in the fluorescence microscopy it equals Itot(t)=3Imag(t), where Imag(t) represents the fluorescence intensity detected at thetamag corresponding to a particular NA/n value. If the correct (true) decay of Itot(t) is substituted into the denominator in the expression for the emission anisotropy r(t), r(t) is a (multi)exponential function of time and it accounts for the high-aperture excitation-detection conditions.

Fluorescence polarization spectroscopy at combined high-aperture excitation and detection: application to one-photon-excitation fluorescence microscopy.

A problem of the one-photon-excitation fluorescence polarization spectroscopy of macroscopically isotropic media, in the case of combined high-aperture excitation and detection, is considered and described in a spherical representation. The case of inhomogeneous intensity distribution in the cross-section of the parallel beam of exciting light, which is converted by an objective lens into inhomogeneous radial distribution of the intensity of the focused exciting light, is also taken into account. The obtained formalism is adapted to the description of confocal fluorescence polarization microscopy. It is shown that the total and magic-angle-detected fluorescence decays do not solely represent the kinetic evolution of the excited-state because of the contribution of the dynamic evolution of photoselected fluorophores. The time-evolution of emission anisotropy is nonexponential. The outlined theory predicts that the total and magic-angle-detected fluorescence decays solely represent the kinetic fluorescence decay, and thereby, the emission anisotropy becomes an (multi)exponential function of time for the excitation-detection cone half-angles not higher than about 15-20 degrees. The initial values of the emission anisotropy are not modified by the application of the excitation-detection apertures if the cone half-angles do not exceed 10-15 degrees. The histograms of unpolarized fluorescence, calculated from the parallel and the perpendicular components of polarized fluorescence, detected at the excitation-detection cones wider than about 65 degrees solely represent the kinetic fluorescence decay. At such conditions, the microscope objective operates like an "integrating sphere". The calibration method, which is based on a general (symmetry adapted) formula describing fluorescence polarization experiments on macroscopically isotropic samples, is discussed. This method enables the analysis of all fluorescence polarization experiments without the necessity of considering the expressions for polarized fluorescence decays relating to a particular experimental case of interest. With this method, any commercially available microscope objective can be calibrated, and its optical properties can be precisely verified. The application of the outlined theory to different fluorescence spectroscopy techniques is indicated. The expressions derived for confocal fluorescence polarization microscopy can be employed in the numerical analysis of the data recovered from the photochemical bioimaging.

Combined genetic algorithm and multiple linear regression (GA-MLR) optimizer: Application to multi-exponential fluorescence decay surface.

The optimization approach based on the genetic algorithm (GA) combined with multiple linear regression (MLR) method, is discussed. The GA-MLR optimizer is designed for the nonlinear least-squares problems in which the model functions are linear combinations of nonlinear functions. GA optimizes the nonlinear parameters, and the linear parameters are calculated from MLR. GA-MLR is an intuitive optimization approach and it exploits all advantages of the genetic algorithm technique. This optimization method results from an appropriate combination of two well-known optimization methods. The MLR method is embedded in the GA optimizer and linear and nonlinear model parameters are optimized in parallel. The MLR method is the only one strictly mathematical "tool" involved in GA-MLR. The GA-MLR approach simplifies and accelerates considerably the optimization process because the linear parameters are not the fitted ones. Its properties are exemplified by the analysis of the kinetic biexponential fluorescence decay surface corresponding to a two-excited-state interconversion process. A short discussion of the variable projection (VP) algorithm, designed for the same class of the optimization problems, is presented. VP is a very advanced mathematical formalism that involves the methods of nonlinear functionals, algebra of linear projectors, and the formalism of Fréchet derivatives and pseudo-inverses. Additional explanatory comments are added on the application of recently introduced the GA-NR optimizer to simultaneous recovery of linear and weakly nonlinear parameters occurring in the same optimization problem together with nonlinear parameters. The GA-NR optimizer combines the GA method with the NR method, in which the minimum-value condition for the quadratic approximation to chi(2), obtained from the Taylor series expansion of chi(2), is recovered by means of the Newton-Raphson algorithm. The application of the GA-NR optimizer to model functions which are multi-linear combinations of nonlinear functions, is indicated. The VP algorithm does not distinguish the weakly nonlinear parameters from the nonlinear ones and it does not apply to the model functions which are multi-linear combinations of nonlinear functions.

Spectroscopic studies into the influence of UV radiation on elastin hydrolysates in water solution.

An investigation into the influence of UV irradiation on elastin hydrolysates dissolved in water was carried out using UV-Vis spectroscopy and spectrofluorometry. It was found that the absorption of elastin hydrolysates in solution increased during irradiation of the sample. For fluorescence of elastin hydrolysates we observed both, a decrease and increase of this value during irradiation of the sample. After UV irradiation of the elastin solution we observed a minor increase of overall absorption, most notably between 250 nm and 280 nm. Moreover, after UV irradiation a wide peak emerged between 290 nm and 310 nm with maximum at about 305 nm. The new peak suggests that new photoproducts are formed during UV irradiation of elastin hydrolysates. The fluorescence of elastin hydrolysates was observed at 305 nm and at 380 nm after excitation at 270 nm. UV irradiation caused fluorescence fading at 305 nm and 380 nm. After 30 min of irradiation a new broad weak band of fluorescence, attributable to new photoproducts, emerged in the UV wavelength region with emission maximum between 400 nm and 500 nm.