Characterization of dissolved organic matter released from Microcystis aeruginosa.

The contribution of dissolved organic matter (DOM) released from phytoplankton (Microcystis aeruginosa) during cultivation and biodegradation was examined to clarify the causes of the organic pollution of Lake Biwa. Two peaks, peak 2 (retention time (RT) = 32 min) and peak 3 (RT = 35 min), were detected in the algal DOM released from Microcystis aeruginosa during cultivation and biodegradation by gel chromatography with a fluorescence detector (Ex = 340 nm, Em = 435 nm). As these peaks correspond with the peaks detected in the surface water of Lake Biwa, one can conclude that the algal DOM released from Microcystis aeruginosa during cultivation and biodegradation makes a considerable contribution to the refractory organic matter in Lake Biwa. Three fluorescence maxima were observed in the cultivation of Microcystis aeruginosa: a fulvic-like fluorescence peak (peak A) with Ex/Em values of 320/430 nm, a protein-like fluorescence peak (peak C) with Ex/Em values of 280/360 nm, and another peak with Ex/Em values of 240/370 nm. The fluorescence material of peak C has a larger MW than that of peak A. The algal-derived DOM from Microcystis aeruginosa has similar fluorescence to fulvic acid of soil origin but exhibits mainly hydrophilic characteristics. In the biodegradation of Microcystis aeruginosa, a fulvic-like fluorescence peak (peak B) with Ex/Em values of 250/440 nm and a peak with Ex/Em values of 320/380 nm were observed.     

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 AND MICROSCOPIC CHARACTERISTICS OF HUMAN SKIN AUTOFLUORESCENCE EMISSION

To improve the understanding of human skin autofluorescence emission, the spectroscopic and microscopic characteristics of skin autofluorescence were studied using a combined fluorescence and reflectance spectroanalyzer and a fiber optic microspectrophotometer. The autofluorescence spectra of in vivo human skin were measured over a wide excitation wavelength range (350–470 nm). The excitation–emission matrices of in vivo skin were obtained. An excitation–emission maximum pair (380 nm, 470 nm) was identified. It was revealed that the most probable energy of skin autofluorescence emission photons increases monotonically and near linearly with increasing excitation photon energy. It was demonstrated that the diffuse reflectance, R, can be used as a first order approximation of the fluorescence distortion factor f to correct the measured in vivo autofluorescence spectra for the effect of tissue reabsorption and scattering. The microscopic in vitro autofluorescence properties of excised skin tissue sections were examined using 442 nm He–Cd laser light excitation as an example. It was demonstrated that the fluorophore distribution inside the skin tissue is not uniform and the shapes of the autofluorescence spectra of different anatomical skin layers vary. The result of this study confirms that the major skin fluorophores are located in the dermis and provides an excellent foundation for Monte Carlo modeling of in vivo autofluorescence measurements.     

Laser-induced fluorescence microscopic system using an optical parametric oscillator for tunable detection in microchip analysis

A laser-induced fluorescence microscopic system based on optical parametric oscillation has been constructed as a tunable detector for microchip analysis. The detection limit of sulforhodamine B (Ex. 520 nm, Em. 570 nm) was 0.2 mol, which was approximately eight orders of magnitude better than with a conventional fluorophotometer. The system was applied to the determination of fluorescence-labeled DNA (Ex. 494 nm, Em. 519 nm) in a microchannel and the detection limit reached a single molecule. These results showed the feasibility of this system as a highly sensitive and tunable fluorescence detector for microchip analysis.     

Shedding light on the laser wavelength effect in Raman analysis of skin epidermises

Confocal Raman microspectroscopy is a promising technique which enables measuring the molecular composition of the skin layers, non-destructively and without extrinsic markers. The Raman approach is increasingly used in skin research but with various experimental conditions. In addition to the different skin types, one of the varying parameters is the wavelength of laser excitation. This parameter contributes strongly in the skin Raman response. The present work aimed to evaluate this effect for 3 different wavelengths, 532, 633 and 785 nm, on pig ear skin models. The Raman signal was assessed in the spectral fingerprint region. According to the Raman response for stability, repeatability, variability and fluorescence contribution, the 785 nm excitation wavelength was shown to be the most suitable for epidermis depth profiling in the fingerprint region.     

Fluorescence excitation spectroscopy for the measurement of epidermal proliferation.

Fluorescence excitation spectroscopy was used to assess cellular turnover in human skin by monitoring changes of endogenous fluorescence. Epidermal proliferation was induced with alpha-hydroxy acids. Commercially available glycolic acid creams (8 and 4% wt/wt concentration) and a vehicle cream (placebo) were applied in a randomized double blinded fashion on subjects' forearms, twice daily for 21 days. Excitation spectra were recorded (excitation 250-360 nm, emission 380 nm) at days 0, 1, 3, 7, 10, 11, 14, 17 and 21. The 295 nm excitation band (assigned to tryptophan moieties) was used in this study as a marker for cellular proliferation. To further reduce the day-to-day variability of the skin fluorescence the intensity of the 295 nm band was normalized to the 334 nm band (assigned to collagen crosslinks). The fluorescence emission intensity from placebo-treated skin remained practically unchanged over the period of the measurements while the fluorescence intensity measured from the glycolic acid-treated skin increased monotonically with treatment. The rate of increase of the excitation intensity with treatment was found to be dose dependent. The epidermal 295 nm band may be used as a quantitative marker to monitor the rate of proliferation of epidermal keratinocytes noninvasively.     

Dual wavelength excitation fluorescence detector for capillary electrophoresis using a pulsed bi-colour light emitting diode

A simple and novel two-colour fluorescence detector for capillary electrophoresis was created using a single bi-colour light emitting diode (LED), multi-band pass excitation and emission filters and a single detector. Excitation light from a blue/red (470/635 nm) bi-colour LED was filtered through a 390/482/563/640 nm multi-band bandpass filter, with emitted light filtered through a 446/523/600/677 nm multi-band bandpass filter before being detected using a photon counting detector. Sequential pulsing of the blue/red LED and deconvolution of the collected fluorescence data allowed extracted electropherograms to be obtained corresponding to excitation with the blue and red LEDs. Optimisation of the pulsed LED conditions revealed an optimum LED on-time of 50 ms, off-time of 30 ms with a pulsed current of 40 mA, giving an effective data acquisition rate of 6.25 Hz. The characteristics of this system were validated by the simultaneous separation and determination of six fluorescent dyes: fluorescein, FITC, coumarin 334, dibromo(R)fluorescein (Ex/Em 470/525 nm), and Cy 5 and the Agilent Bioanalyser DNA dye (Ex/Em 635/670 nm). Under optimum conditions, the detection limits for FITC, fluorescein and Cy 5 were 69 nM, 42 nM and 289 nM (S/N = 3), respectively. These were lower than those obtained with continuous operation of the individual wavelengths at a constant current of 20 mA, but were slightly higher than those obtained using dedicated single wavelength filter combinations designed specifically for use with these fluorophores. The intraday repeatability (n = 6) of migration times was less than 1.0% and less than 3.4% for peak areas, while interday (n = 3) migration time and peak area reproducibility were less than 0.9% and 3.6%, respectively. This simple detector is capable of performing quantitative two-wavelength excitation without the need for complex optics and light source configurations.     

Photodynamic Imaging of a Rat Pancreatic Cancer with Pheophorbide a

Laser-induced fluorescence of pheophorbide a (Ph- a ) was used for in vitro photodynamic imaging (PDI) of a rat pancreatic acinar tumor. A 400 nm excitation induced a 470 nm autofluorescence and a 678 nm dye fluorescence in tumors and their surrounding pancreas 24 h after a 9 mg kg⁻¹ body weight Ph- a intravenous administration. With lower intensities in these blood-rich tumors than in pancreas, Ph- a fluorescence signals are unable to provide tumor images. A dimensionless function (the ratio of Ph- a fluorescence by autofluorescence, called R_t for the tumor and R_p for the pancreas) was used for fluorescence contrast calculation (C = R_t/R_p) between six tumors and their paired pancreas. Among five available laser excitation wavelengths, only the 355 nm excitation gave a distinctive contrast (C = 1.5). The PDI of six intrapancreatic tumors and their intraperitoneal metastasis and of two control normal pancreas was thus performed ex vivo using a 355 nm excitation source delivered by a tripled Nd: YAG laser and a charged-coupled device camera. Fluorescence images were recorded at 680 nm (dye), 640 nm (background) and 470 nm (autofluorescence) through three corresponding 10 nm width bandpass filters. Computed division for each pixel of Ph- a fluorescence values by autofluorescence generated false color image. In this way, contrasted tumor images were obtained. But in five out of six animals false-positive images were present due to an autofluorescence decrease in some normal pancreatic areas. A 470 nm autofluorescence imaging on the same tumors gave in all cases false-positive image and false-negative in half of the cases. These observations suggest that autofluorescence alone is unable to achieve accurate PDI of pancreatic carcinoma and that using Ph- a as a PDI dye needs strong improvements.     

Development of a hypoxia-selective near-infrared fluorescent probe for non-invasive tumor imaging

A near-infrared fluorochrome, GPU-311, was designed, synthesized and evaluated for its application in non-invasive imaging of tumor hypoxia. Efficient synthesis was achieved by nucleophilic substitution and click chemistry ring using the bifunctional tetraethylene glycol linker 2 containing thiol and azide groups for the conjugation of the propargylated nitroimidazole 1 and the heptamethine cyanine dye 3 bearing a 2-chloro-1-cyclohexenyl ring. GPU-311 exhibited long excitation and emission wavelength (Ex/Em=785/802?nm) and a decent quantum yield (0.05). The water solubility and hydrophilicity of GPU-311 increased. After in vitro treatment of SUIT-2/HRE-Luc pancreatic cancer cells with GPU-311, a higher level of fluorescence was observed selectively in hypoxia than in normoxia. However, in vivo fluorescence imaging of a mouse xenograft model after GPU-311 administration revealed inadequate accumulation of GPU-311 in tumors due to its rapid elimination through the liver.     

Glucose-dependent changes in NAD(P)H-related fluorescence lifetime of adipocytes and fibroblasts in vitro: potential for non-invasive glucose sensing in diabetes mellitus

The aim of this study was to test the hypothesis that glucose can be monitored non-invasively by measuring NAD(P)H-related fluorescence lifetime of cells in an in vitro cell culture model. Autofluorescence decay functions were measured in 3T3-L1 adipocytes by time-correlated single-photon counting (excitation 370nm, emission 420-480nm). Free NADH had a two-exponential decay but cell autofluorescence fitted best to a three-exponential decay. Addition of 30mM glucose caused a 29% increase in autofluorescence intensity, a significantly shortened mean lifetime (from 7.23 to 6.73ns), and an increase in the relative amplitude and fractional intensity of the short-lifetime component at the expense of the two longer-lifetime components. Similar effects were seen with rotenone, an agent that maximizes mitochondrial NADH. 3T3-L1 fibroblasts stained with the fluorescent mitochondrial marker, rhodamine 123 showed a 16% quenching of fluorescence intensity when exposed to 30mM glucose, and an increase in the relative amplitude and fractional intensity of the short lifetime at the expense of the longer lifetime component. We conclude that, though the effect size is relatively small, glucose can be measured non-invasively in cells by monitoring changes in the lifetimes of cell autofluorescence or of a dye marker of mitochondrial metabolism. Further investigation and development of fluorescence intensity and lifetime sensing is therefore indicated for possible non-invasive metabolic monitoring in human diabetes.     

The application of a compact multispectral imaging system with integrated excitation source to in vivo monitoring of fluorescence during topical photodynamic therapy of superficial skin cancers

A novel, compact and low-cost multispectral fluorescence imaging system with an integrated excitation light source is described. Data are presented demonstrating the application of this method to in vivo monitoring of fluorescence before, during and after topical 5-aminolevulinic acid photodynamic therapy of superficial skin cancers. The excitation source comprised a fluorescent tube with the phosphor selected to emit broadband violet light centered at 394 nm. The camera system simultaneously captured spectrally specific images of the fluorescence of the photosensitizer, protoporphyrin IX, the illumination profile and the skin autofluorescence. Real-time processing enabled images to be manipulated to create a composite image of high contrast. The application and validation of this method will allow further detailed studies of the characteristics and time-course of protoporphyrin IX fluorescence, during topical photodynamic therapy in human skin in vivo.     

Selective determination of cysteines through precolumn double-labeling and liquid chromatography followed by detection of intramolecular FRET

In this paper we introduce a novel approach for highly selective and sensitive analysis of cysteines (glutathione, cysteine, and homocysteine). This method is based on the detection of intramolecular fluorescence resonance energy transfer (FRET) in a liquid chromatography (LC) system after double-labeling of the amino and sulfhydryl groups of the cysteines. In this detection process, we monitored the FRET between the amine-derivatized and thiol-derivatized fluorophores. We screened 16 combinations of fluorescent reagents. As a result, FRET occurred most effectively when the sulfhydryl and amino groups of the cysteines were derivatized with 7-diethylamino-3-[{4'-(iodoacetyl)amino}phenyl]-4-methylcoumarin (DCIA, Ex/Em 390/480 nm) and 4-fluoro-7-nitrobenz-2-oxo-1,3-diazole (NBD-F, Ex/Em 480/540 nm), respectively, in this order. The double-labeled cysteines emitted NBD-F fluorescence (540 nm) through an intramolecular FRET process when they were excited at the wavelength of maximum excitation of DCIA (390 nm). The generation of FRET was confirmed by comparison with analysis of n-amylamine or tryptophan (amines without a sulfhydryl group) and 6-mercaptohexanol (thiol without an amino group) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the double-labeled cysteines (DCIA and NBD-F) when performing LC on an ODS column with isocratic elution. The limits of quantification (signal-to-noise ratio = 10) and detection (signal-to-noise ratio = 3) for the cysteines, for a 20-μL injection volume, were in the range 150-670 fmol and 46-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of a system which takes advantage of conventional detection of the derivatives. Furthermore, this method provides sufficient selectivity and sensitivity to determine the total cysteines present in the plasma of healthy humans.     

Spectrofluorometric determination of kynurenic acid with horseradish peroxidase in the presence of hydrogen peroxide.

The catalytic activity of horseradish peroxidase (HRP) in the presence of hydrogen peroxide has been investigated for the fluorescent derivatization of kynurenic acid under conditions with no exposure to light. Non-fluorescent kynurenic acid was converted into a fluorescent compound (Ex: 367 nm, Em: 470 nm) with HRP in the presence of hydrogen peroxide, and the optimum conditions of this fluorescent derivatization were investigated. Moreover, this fluorescent derivatization was developed for a spectrofluorometric determination of trace amounts of kynurenic acid by measuring the fluorescence intensity of the fluorescent compound. The calibration curve obtained was linear from 1.0 to 10.0 nmol of kynurenic acid in a 1.0 mL sample solution. The relative standard deviation at 5.0 nmol of kynurenic acid was 5.71% (n=5). By adjusting the bandwidths for both the excitation and emission to 15 nm, the calibration curve was also linear in the range between 0.1 to 1.0 nmol of kynurenic acid in a 1.0 mL sample solution. This method was applied to the fluorometric determination of trace amounts of kynurenic acid in the control sera.     

The effect of UV absorbing sunscreens on the reflectance and the consequent protection of skin

The in vivo reflectance spectra of Caucasian skin, coated with preparations containing sunscreen vehicle, vehicle with olive oil and vehicle with the UVB and UVA absorbers 2-ethylhexyl-4-methoxycinnamate and 4-t-butyl-4'-methoxydibenzoylmethane were determined. All preparations reduced the reflectance of skin throughout the UVA spectral range (320 to 400 nm), with the sunscreen preparations containing the UVB and UVB plus UVA absorbers reducing the reflectance more than the sunscreen vehicle alone. This phenomenon, which facilitates the penetration of UV radiation to the lower epidermis and dermal layers of skin and therefore lessens sunscreen efficacy, is attributed to optical coupling mediated by refractive index matching of the sunscreen to the upper epidermis. The greater reduction in skin diffuse reflectance caused by sunscreens containing methoxycinnamate is associated with this compound's high refractive index. Also, by determining the excitation spectra of the autofluorescence originating from the dermal layer of skin, the transmission spectra of the various components of sunscreen on skin were established, and these were in good general agreement with previously published spectra.     

Spectrally resolved fluorescence imaging of human colonic adenomas

Native fluorescence (autofluorescence) of human tissues can be a valuable source of diagnostic information for detecting premalignant and malignant lesions in the human body. Digital imaging of autofluorescence may be useful for localization of such lesions during endoscopic examinations. Tissue fluorescence of 31 adenomatous polyps obtained from 16 patients has been excited in vitro using the 325 nm line of a He-Cd laser. Digital images of the autofluorescence are recorded in six spectral bands. This study provides new data about the spatial distributions of autofluorescence intensities emitted in different spectral bands by colonic adenomatous lesions and normal colonic mucosa. Areas characterized by autofluorescence intensity lower than in normal mucosa are found for a majority of the polyps under study. The observed patterns of spatial distribution differ for the different spectral bands and for different polypoid lesions. No inverse correlation is found between the emission intensity and the thickness of colonic mucosa. The results indicate the spectral bands most useful for diagnostic applications and demonstrate the complexity of the optical processes involved in shaping both the spectra and intensities of the autofluorescence.     

Dependence of fibroblast autofluorescence properties on normal and transformed conditions. Role of the metabolic activity

The dependence of autofluorescence properties on the metabolic and functional engagement and on the transformation condition was studied on single cells. Normal Galliera rat fibroblasts at low subculture passage (cell strain), at high subculture passage (stabilized cell line), and transformed cell line derived from a rat sarcoma were used as a cell model. The study was performed by microspectrofluorometric and fluorescence imaging technique. The autofluorescence properties of cells were studied by excitation at two wavelengths, namely 366 nm and 436 nm, that are known to favor the emission of different fluorophores. Spectral shape analysis indicated that under excitation at 366 nm autofluorescence is ascribable mainly to coenzyme molecules, particularly to reduced pyridine nucleotides, while under excitation at 436 nm, flavin and lipopigment emission is favored. The energetic metabolic engagement of the different cell lines was analyzed in terms both of parameters related to anaerobic-aerobic pathways (biochemical assay) and of mitochondrial features (supravital cytometry). The results showed that the cell strain and the stabilized and transformed cell lines can be distinguished from one another on the basis of both overall fluorescence intensity and the relative contributions of spectral components. These findings indicated a relationship between autofluorescence properties and energetic metabolism engagement of the cells that, in turn, is dependent on the proliferative activity and the transformed condition of the cells. In that it is a direct expression of the energetic metabolic engagement, autofluorescence can be assumed as an intrinsic parameter of the cell biological condition, suitable for diagnostic purposes.     

Fluorometric Determination of Iron(Iii) with O-Hydroxyhydroquinonephthalein in the Presence of Brij 58

Abstract

A highly sensitive fluorescence reaction of iron(III) with o-hydroxyhydroquinonephthalein (Qnph) in the presence of various surfactants, and its application to the fluorophotometry of trace amounts of iron(III) is described. the method is based on the fluorescence quenching reaction between Qnph and iron(III) in the presence of Brij 58 at pH 3–4. the quenching calibration graph was linear over the range 0 – 300 ng per ml iron(III) by using fluorescence reaction at Em 525 nm with Ex 470 nm, and the iron(III) detection limit was 5 ng/ml. the proposed method is simple, rapid and does not involve heating or solvent extraction.     

A study of Na2 diffuse bands in violet by the excitation through self-broadenedD-lines

Dye-laser, tuned over the self-broadened wings of the sodiumD-lines, was used for the excitation of sodium dimer diffuse bands in the region from 400 nm to 455 nm. The fluorescence and selected excitation spectra for various spectral phenomena at different experimental conditions have been obtained and analyzed. The models for the qualitative explanation of the excitation mechanism are discussed.     

In vivo Laser-induced Fluorescence Imaging of a Rat Pancreatic Cancer with Pheophorbide-a

Laser-induced fluorescence (LIF) of pheophorbide-a (Ph-a) was used for imaging of a rat pancreatic tumor. Using a dimensionless function (the ratio of Ph-a fluorescence by bluish autofluorescence), the fluorescence contrasts between excised tumors and their paired pancreas were investigated up to 48 h after a 9 mg kg^-1 Ph-a intravenous administration. Among five tested excitation wavelengths, 355 and 610 nm excitations gave the best distinctive contrasts, both 48 h after dye injection. The LIF imaging of six intrapancreatic tumors and six healthy pancreas was carried out in vivo using two laser excitations: 355 nm (Nd:YAG + tripling) for bluish autofluorescence and 610 nm (rhodamine 6G dye) for reddish autofluorescence and dye emission. Images were recorded through bandpass filters at 470 and 640 nm (autofluorescence) and at 680 nm (dye + autofluorescence) with an intensified charged-coupled device camera. Autofluorescence as Ph-a fluorescence images did not allow accurate LIF diagnosis of pancreatic carcinoma. An image processing, including for each pixel a computed division of Ph-a fluorescence (after subtraction of reddish autofluorescence) by bluish autofluorescence intensity generated poorly contrasted tumor images in five of six and false tumor localization in one of three of the tumor-bearing pancreas. A fitting of the digital 640 nm autofluorescence up to the mean 680 nm fluorescence intensity in pancreas prior to subtraction allowed a safe diagnosis to be made with well-contrasted tumor images. To assess automation ability of the processing, a same fitting coefficient (mean of individual values) was applied. In this way, false-negative (one of six) and false-positive (two of six) images were present in tumor-bearing animals as false-positive in one-half of the controls. A successful standardized procedure was then applied with a normalization of 640 and 680 nm pancreas intensities to a same set threshold prior processing. In opposition to thin-layered hollow organs, such as bronchial tube or digestive tract, LIF imaging of carcinoma inserted in a compact organ is exhausting. The use of a dye excitable in the red wavelength range (610 nm for Ph-a) may partly solve this problem, rendering LIF imaging more accurate and potentially automated.     

Effects of photodynamic therapy with topical application of 5-aminolevulinic acid on normal skin of hairless guinea pigs.

Photodynamic therapy (PDT) is a relatively new approach to the treatment of neoplasms which involves the use of photoactivatable compounds to selectively destroy tumors. 5-Aminolevulinic acid (ALA) is an endogenous substance which is converted to protoporphyrin IX (PpIX) in the synthetic pathway to heme. PpIX is a very effective photosensitizer. The goal of this study was to evaluate the effect of PDT using topical ALA on normal guinea pig (g.p.) skin and g.p. skin in which the stratum corneum was removed by being tape-stripped (TS). Evaluation consisted of gross examination, PpIX fluorescence detection, reflectance spectroscopy, and histology. There was no effect from the application of light or ALA alone. Normal non-TS g.p. skin treated with ALA and light was unaffected unless high light and ALA doses were used. Skin from which the stratum corneum was removed was highly sensitive to treatment with ALA and light: 24 h after treatment, the epidermis showed full thickness necrosis, followed by complete repair within 7 d. Time-dependent fluorescence excitation and emission spectra were determined to characterize the chromophore and to demonstrate a build-up of the porphyrin in the skin. These data support the view that PDT with topical ALA is a promising approach for the treatment of epidermal cutaneous disorders.