Fluorescence Of Fluorophore Research Articles

Design and use of fluorogenic aldehydes for monitoring the progress of aldehyde transformations.

We describe the first examples of fluorogenic aldehydes useful for monitoring many types of reactions including aldol reactions, allylations, and reductions. The fluorogenic aldehydes were constructed by covalent combination of a fluorophore and an aldehyde moiety via a linker. In the resulting single molecule, the aldehyde functioned as a quencher of the fluorophore's fluorescence. The reaction product, modified at the aldehyde functionality, no longer served as an effective quencher. The reaction products showed up to approximately 80-fold higher fluorescence than the aldehyde reactants.

Measurement of fluorophore concentrations and fluorescence quantum yield in tissue-simulating phantoms using three diffusion models of steady-state spatially resolved fluorescence

Steady-state diffusion theory models of fluorescence in tissue have been investigated for recovering fluorophore concentrations and fluorescence quantum yield. Spatially resolved fluorescence, excitation and emission reflectance were calculated by diffusion theory and Monte Carlo simulations, and measured using a multi-fibre probe on tissue-simulating phantoms containing either aluminium phthalocyanine tetrasulfonate (AlPcS4), Photofrin® or meso-tetra-(4-sulfonatophenyl)-porphine dihydrochloride (TPPS4). The accuracy of the fluorophore concentration and fluorescence quantum yield recovered by three different models of spatially resolved fluorescence were compared. The models were based on: (a) weighted difference of the excitation and emission reflectance, (b) fluorescence due to a point excitation source or (c) fluorescence due to a pencil beam excitation source. When literature values for the fluorescence quantum yield were used for each of the fluorophores, the fluorophore absorption coefficient (and hence concentration) at the excitation wavelength (μa,x,f) was recovered with a root-mean-square accuracy of 11.4% using the point source model of fluorescence and 8.0% using the more complicated pencil beam excitation model. The accuracy was calculated over a broad range of optical properties and fluorophore concentrations. The weighted difference of reflectance model performed poorly, with a root-mean-square error in concentration of about 50%. Monte Carlo simulations suggest that there are some situations where the weighted difference of reflectance is as accurate as the other two models, although this was not confirmed experimentally. Estimates of the fluorescence quantum yield in multiple scattering media were also made by determining μa,x,f independently from the fitted absorption spectrum and applying the various diffusion theory models. The fluorescence quantum yields for AlPcS4 and TPPS4 were calculated to be 0.59 ± 0.03 and 0.121 ± 0.001 respectively using the point source model, and 0.63 ± 0.03 and 0.129 ± 0.002 using the pencil beam excitation model. These results are consistent with published values.

Recovery of turbidity free fluorescence from measured fluorescence: an experimental approach

Fluorescence from fluorophores embedded in a turbid medium like biological tissue gets strongly modulated by the wavelength dependent absorption and scattering properties of tissue. This makes it extremely difficult to extract valuable biochemical information from tissue which is present in the intrinsic line shape and intensity of fluorescence from tissue fluorophores. We present an experimental approach to remove the distorting effect of scattering and absorption on intrinsic fluorescence of fluorophores embedded in a turbid medium like tissue. The method is based on simultaneous measurement of polarized fluorescence and polarized elastic scattering spectra from a turbid medium. The polarized fluorescence normalized by the polarized elastic scattering spectra (in the wavelength range of fluorescence emission) was found to be free from the distorting effect of absorption and scattering properties of the medium. The applicability range of this technique to recover intensity and line shape information of intrinsic fluorescence has been investigated by carrying out studies on a variety of tissue phantoms having different absorption and scattering properties. The results obtained show that this technique can be used to recover intrinsic line shape and intensity information of fluorescence from fluorophores embedded in a scattering medium for the range of optical transport parameters typically found in biological tissue.

Tripodal ligand incorporating dual fluorescent ionophore: a coordinative control of photoinduced electron transfer.

p-(N,N-Dimethylamino)benzenesulfonamide (DMABSA), a dual fluorescent fluorophore, has been derivatized into two new fluoroionophores for transition metal cations. The electron-acceptor sulfonamide group has been N-substituted by a 2-pyridylmethylene group to lead to a bidentate ligand which forms a 2:1 complex with Cu(II). The crystal structure of the copper(II) complex is reported. The Cu(II) is coordinated through the pyridine N- and sulfonamide N-deprotonated atom which account for the blue-shift of the absorption and the partly quenched fluorescence. When DMABSA was incorporated into the tris(2-aminoethyl)amine (tren), a tripodal, dual-fluorescent ligand, is obtained which shows higher binding affinity for Zn(II) than for Cu(II). Furthermore, the large increase of the short wavelength emission and the disappearance of the TICT emission, upon Zn complexation, allow measurements of the Zn(II) concentration from relative fluorescence intensity at two wavelengths.

The Fluorosome™ technique for investigating membrane on- and off-loading of drugs by β-CD and sonicated SUV

The application of the Fluorosome technique to test drug delivery systems is described. Fluorosomes, egg phosphatidylcholine liposomes with bilayer embedded fluorophores, were employed to investigate the ability of sonicated small unilamellar vesicles (sSUV) and β-cyclodextrins (β-CD) to deliver drugs into or extract drugs from the fluorosome’s phospholipid bilayer. The addition of phloretin to a fluorosome suspension resulted in fluorescence reduction reflecting phloretin entering the bilayer and quenching fluorophore fluorescence. Subsequent addition of sSUV to phloretin pretreated fluorosomes showed an increase in fluorescence reflecting phloretin extraction from the fluorosome membrane. Sequential additions of β-estradiol loaded β-CD to fluorosomes as well as the addition of β-estradiol alone resulted in fluorescence reduction due to β-estradiol insertion into the membrane. Further addition of pure β-CD resulted in a fluorescence increase indicating β-estradiol extraction from the fluorosome membrane.

The synthesis and evaluation of o-phenylenediamine derivatives as fluorescent probes for nitric oxide detection

A series of molecular probes for the determination of nitric oxide (NO) have been prepared. Each probe consists of an anthracene, coumarin or acridine fluorophore coupled to an electron rich o-phenylenediamine group. The o-phenylenediamine group can be substituted with methyl or methoxy groups to enhance its electron rich nature. The fluorophore fluorescence is quenched by photoelectron transfer (PET) from the aromatic amine to the lowest unoccupied orbital of the excited state fluorophore. Reaction with nitrosating species converts the o-phenylenediamine group into an electron deficient benzotriazole derivative. This group has a higher oxidation potential and does not quench the fluorophore fluorescence by photoelectron transfer so that these products are highly fluorescent. Some benzotriazole derivatives were made preparatively by alternative synthetic routes. The formation of fluorescent probes was evaluated by treatment of the precursors with nitrous fumes and S-nitroso-N-acetylpenicillamine (SNAP).

A Preparative and Spectroscopic Study of Fluorophores for Zinc(II) Detection

Zinc(II) specific fluorophores are of substantial importance in the study of intracellular Zn 2+ . Two such widely used fluorophores are 2-methyl-8-(4-toluenesulfonamido)-6-quinolyloxyacetic acid, Zinquin (1), and its ethyl ester, Zinquin ester (2), which fluoresce strongly when bound by Zn 2+ . To gain insight into the factors affecting the fluorescence of such fluorophores the closely related 4-methyl-N-(6-methoxy-2-methyl-8-quinolyl)-benzenesulfonamide (3), and nine analogues (4–12), substituted at sulfur by nine different substituents have been prepared and their fluorescing characteristics and those of their Zn 2+ complexes have been examined. The nine substituents are: 2,2,2-trifluoroethyl (4), 4-methoxyphenyl (5), 4-acetamidophenyl (6), 4-bromophenyl (7), 4-nitrophenyl (8), 3-trifluoromethylphenyl (9), naphth-1-yl (10), naphth-2-yl (11) and 5-dimethylaminonaphth-1-yl (12). Under neutral conditions in 75/25% v/v ethanol/water solutions, (3)–(7) and (9)–(11) fluoresce weakly in the free state, (8) does not fluoresce and (12) fluoresces strongly. Ultraviolet (UV)-visible spectroscopy shows that (3)–(12) complex to Zn 2+ , but unlike the remainder, the complexes of (8) and (12) do not fluoresce, with those possessing electron-withdrawing substituents, (4) and (9), being the most fluorescent. On this basis ethyl-2-(2-methyl-quinolyloxy-8-(2,2,2-trifluoroethylsulfonamido))acetate (19) was prepared and its Zn 2+ complex was found to be substantially more fluorescent than that of (2).

Flow Cytometry-Based Biosensor for Detection of Multivalent Proteins

Microsphere-based flow cytometric detection of cholera toxin (CT) through distance-dependent fluorescence resonant energy transfer (FRET) has been developed. Simultaneous double-fluorescence changes induced by multivalent interactions between CT and fluorophore (both fluorescence donor and acceptor)-labeled ganglioside GM1 on a biomimetic membrane surface (supported bilayers of phospholipids) can be measured by a commercial flow cytometer, providing a convenient and sensitive detection method for CT. The flow cytometry-based biosensor is capable of detecting less than 10 pM CT within 30 min. The signal generation strategy coupled with flow cytometry also provides a convenient method for kinetic studies of multivalent interactions. The surface density and the ratio of donor/acceptor-labeled GM1 on the surfaces of phospholipid bilayers are optimized to achieve high sensitivity.

Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry

We report a novel method to quantify fluorescence intensity and lifetime of free (solution) and particle-bound fluorophore in a flow stream. The technique combines flow cytometry and frequency-domain lifetime spectroscopy principles to make unique fluorescence measurements on free fluorophore and fluorophore-labeled particles. Fluorophore-labeled microspheres suspended in a fluorophore solution are analyzed as they flow through a chamber and pass across an intensity-modulated laser beam consisting of a continuous-wave (cw) direct-current (dc) and high-frequency (sine wave) excitation component. Fluorescence emission signals consisting of a dc-offset steady-state sinusoidal signal (fluorophore solution) and a sinusoidally modulated Gaussian-shaped signal pulse (fluorophore-labeled particles) are processed electronically to quantify intensities and lifetimes. The cw-excited, particle-associated fluorophore (pulse) and steady-state (dc) solution fluorescence intensity signals are measured using low-pass filtering to remove the high-frequency signal components and an ac-coupled and a gated dc amplifier to process the respective particle-bound and free fluorophore signals. The high-frequency excited, particle-bound fluorophore and free fluorophore lifetimes are individually measured using two pairs of phase-sensitive detectors to provide signals proportional to the sine and cosine of the respective phase shifts, which are ratioed to determine the respective lifetimes. The fluorescence signal intensity and lifetime detection channel outputs are displayed as frequency distribution histograms using a computer-based data acquisition system.

A new rapid method to detect inhibition of Amadori product generated by δ-gluconolactone

Advanced glycation endproducts (AGEs) have been implicated as a major pathogenic process in leading to diabetic complications. An increasing number of drug candidates have recently been developed as potential inhibitors of AGEs. Aminoguanidine, a hydrazine-like molecule is the first drug that was extensively studied both in vitro and in vivo as an inhibitor of AGE formation. Several assay methods have been proposed to determine the inhibitory effect of glycation inhibitors, including assays based on inhibition of specific fluorescence generated in the course of glycation and AGE-formation; assays based on the inhibition of AGE-protein crosslinks, and dimer and trimer formation; and specific ELISA assays using anti-AGE antibodies for quantitative measurement of AGEs in the presence and absence of the inhibitor. However, none of these assays can accurately evaluate chemical intermediates and products generated during the early stages of glycation. We have devised a new rapid assay method for evaluation of the early stage of glycation (Amadori product). This assay is based on the interaction of δ-gluconolactone (δ-Glu), an oxidized (ketoaldehyde) analogue of glucose, with hemoglobin present in blood samples. The assay involves determination of the percentage of glycated hemoglobin (HbA 1C) after incubation at 37° for 16 h with δ-Glu (50 mmol/L) using a dedicated ion-exchange high-performance liquid chromatography (HPLC) system. The results using normal human red blood cells show HbA 1C levels to be 180% higher than baseline controls. The effects of various inhibitors are determined by measuring the levels of HbA 1C by the compound in question compared to δ-Glu-treated vehicle only blood samples. This new assay provides a relevant and physiological model to study glycation and potential inhibitors. Furthermore, it offers a means to differentiate between inhibitors of the early and late stages of glycation and provides a rapid method of screening large numbers of potential inhibitors of glycation. Contrary to the assay methods, which are based on the measurement of fluorescence of fluorophores generated during glycation, the proposed assay does not suffer from the possible problem of overlapping and interference of AGE-specific fluorescence with the intrinsic fluorescence of the inhibitor compound.

Laser‐Induced Fluorescence (LIF) Recognition of the Structural Composition of Porcine Heart Valves

Abstract— Reconstruction and replacement of heart valves with grafts from pig tissue is a common procedure. However, bioprosthetic valves wear out in a shorter time span than mechanical valves. Bioprosthetic valve structure may contribute to degenerative changes that lead to valve failure. There is, at present, no method to examine the structure of a tissue valve prior to implant. Laser‐induced fluorescence (LIF) of natural fluorophores is an elegant method developed for the detection of tumors, dermal lesions and atherosclerosis. We have studied LIF as a potential diagnostic technique for analysis of valvular tissue. Using excimer laser excitation, we examined natural fluorescence recorded from porcine aortic, mitral and pulmonary valves. All three valve outflow surface tissue layers are less fluorescent at 390–450 nm than the inflow layers. Immunohistochemical analysis of collagen I and elastin content in inflow and outflow surface layers of all three valves correlated well with LIF intensities and dI/dΛ values at selected wavelengths. In conclusion, the differences observed in emitted LIF from valve surface layers are found to correlate well with diversity in the structural protein content. The LIF spectroscopic measurements may provide an appropriate tool for examination of tissue valve structure prior to use for implantation.

Laser-induced fluorescence (LIF) recognition of the structural composition of porcine heart valves.

Reconstruction and replacement of heart valves with grafts fro pig tissue is a common procedure. However, bioprosthetic valves wear out in a shorter time span than mechanical valves. Bioprosthetic valve structure may contribute to degenerative changes that lead to valve failure. There is, at present, no method to examine the structure of a tissue valve prior to implant. Laser-induced fluorescence (LIF) of natural fluorophores is an elegant method developed for the detection of tumors, dermal lesions and atherosclerosis. We have studied LIF as a potential diagnostic technique for analysis of valvular tissue. Using excimer laser excitation, we examined natural fluorescence recorded from porcine aortic, mitral and pulmonary valves. All three valve outflow surface tissue layers are less fluorescent at 390-450 nm than the inflow layers. Immunohistochemical analysis of collagen I and elastin content in inflow and outflow surface layers of all three valves correlated well with LIF intensities and dI/d lambda values at selected wavelengths. In conclusion, the differences observed in emitted LIF from valve surface layers are found to correlate well with diversity in the structural protein content. The LIF spectroscopic measurements may provide an appropriate tool for examination of tissue valve structure prior to use for implantation.

Two-photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm

We report on two-photon fluorescence excitation (TPE) action cross sections for five widely used molecular fluorophores. Measurements were performed by use of ultrashort (~100-fs) Ti:sapphire pulsed excitation over the range 690-960 nm. TPE spectra were obtained by comparison with a fluorescein calibration standard. Large cross sections were found for the cyanine reagent Cy 3 (~140 GM) and for Rhodamine 6G (~150 GM), both at 700 nm [1 GM = 10(-50) (cm(4) s)/photon]. Several fluorophores show interesting and desirable blue shifts with respect to twice the one-photon absorption wavelength. Fluorophore fluorescence intensities showed no significant departure (?4%) from quadratic illumination power dependence, indicating genuine two-photon processes. Implications of these measurements for two-photon laser-scanning microscopy are discussed.

Characterization of native fluorescence from DMBA-treated hamster cheek pouch buccal mucosa for measuring tissue transformation.

The steady-state native fluorescence spectra of extracts of normal mucosa as well as of different stages of oral lesion of the 7,12-dimethylbenz (a) anthracene (DMBA)-induced hamster cheek pouch carcinogenesis model have been measured and analysed at 405 nm excitation. The emission spectra were scanned from 430 to 700 nm to characterize the native fluorescence of endogenous porphyrin and other fluorophores under various tissue transformation conditions, such as hyperplasia, papilloma, early invasive carcinoma and well-differentiated squamous cell carcinoma. Two ratio parameters, R1 = (I530/I620) and R2 = (I530/I630), are introduced to quantify the diagnostic potentiality. The ratio values were found to decrease as the stage of the cancer increases. The suggested critical values for both R1 and R2 for normal mucosa is above three, and the suggested critical value for tissues with lesion is less than three. It was also found that the values for R1 and R2 for well-differentiated squamous cell carcinoma is less than one. The ratio parameter R2 is selected for discrimination between normal mucosa and oral lesions, as the difference in the R2 value between normal and DMBA-treated tissue is higher than that for R1.

Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores: influences of some environmental factors recorded with a confocal laser scanning microscope.

We report on the spectra and fluorescence lifetimes of four commonly used fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine isothiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3). Fluorescence lifetime recordings revealed that these spectrally overlapping fluorophores can be individually detected by their lifetimes, indicating that at least four fluorophores can be individually identified in discrete tissue domains by confocal microscopy. A further advantage of lifetime recordings is that fluorophores that emit light within the same wavelength band can be used and chromatic aberrations are therefore circumvented, thereby improving the spatial accuracy in imaging of multiple fluorophores. Low and high pH, respectively, tended to influence fluorophore emission spectra and fluorescence lifetime. IgG conjugation of the fluorophores tended to shift the spectra towards longer wavelengths and to change the fluorescence lifetimes. The IgG-conjugated form of the fluorophores may, when applied to tissue specimens, change the emission spectrum and lifetime. In addition, different tissue embedding procedures may influence fluorescence lifetime. These observations emphasize the importance of spectral and lifetime characterization of fluorescent probes within the chemical context in which they will be used experimentally. Changes in spectra and fluorescence lifetimes may be a useful tool to gain information about the chemical environment of the fluorophores.

Enhancement of the reductive activation of chloroplast fructose‐1,6‐bisphosphatase by modulators and protein perturbants

To characterize the mechanism of chloroplast fructose-1,6-bisphosphatase activation, we have examined kinetic and structural changes elicited by protein perturbants and reductants. At variance with its well-known capacity for enzyme inactivation, 150 mM sodium trichloroacetate yielded an activatable chloroplast fructose-1,6-bisphosphatase in the presence of 1.0 mM fructose 1,6-bisphosphate and 0.1 mM Ca2+. Other sugar bisphosphates did not replace fructose 1,6-bisphosphate whereas Mg2+ and Mn2+ were functional in place of Ca2+. Variations of the emission fluorescence of intrinsic fluorophores and a noncovalently bound extrinsic probe [2-(p-toluidinyl)naphthalene-6-sulfonate] indicated the presence of conformations different from the native form. A similar conclusion was drawn from the analysis of absorption spectra by means of fourth-derivative spectrophotometry. The effect of these conformational changes on the reductive process was studied by subsequently incubating the enzyme with dithiothreitol. The reaction of chloroplast fructose-1,6-bisphosphatase with dithiothreitol was accelerated 13-fold by the chaotropic anion: second-order rate constants were 48.1 M-1.min-1 and 3.7 M-1.min-1 in the presence and in the absence of trichloroacetate, respectively. Thus, the enhancement of the reductive activation by compounds devoid of redox activity illustrated that the modification of intramolecular noncovalent interactions of chloroplast fructose-1,6-bisphosphatase plays an essential role in the conversion of enzyme disulfide bonds to sulfhydryl groups. In consequence, a conformational change would operate concertedly with the reduction of disulfide bridges in the light-dependent activation mediated by the ferredoxin-thioredoxin system.

Fluctuation in structure of inclusion complexes of cyclodextrins with fluorescent probes

The lifetime distribution of the fluorescence from inclusion complexes of cyclodextrins with anilinonaphthalenesulfonates (ANSs) was analyzed by time-correlated single-photon counting measurements. The very large width of the lifetime distribution as well as the dependence of the apparent maximum wavelength of the fluorescence on the excitation wavelength indicates that the conformation of the excited TNS molecules in cyclodextrin cavities is distributed within a wide range. These observations indicate a striking similarity between the fluorescence of fluorophores in proteins and that in the inclusion complexes.

Dynamic fluorescence of extrinsic fluorophores as a tool for studying protein conformational substates

The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.

Fluorescence Quenching Studies of Near-Infrared Fluorophores

Near-infrared laser dyes were studied with the use of conventional fluorescence quenching. A systematic study of the effects of different quenchers and temperature on the fluorescence of near-infrared fluorophores is presented. Differences in the quenching of near-infrared fluorophores were observed for quenching by ionic quenchers. Quenching with ionic and nonionic quenchers led to nonlinear quenching results.

New methods for spectrofluorometer monochromator wavelength calibration

A method is presented for wavelength calibration of spectrofluorometer monochromators. It is based on the distortion that the characteristic absorption bands of glass filters (holmium or didymium oxide), commonly used for calibration of spectrophotometers, introduce in the emitted fluorescence of fluorophores like indole, diphenyl hexatriene, xylene or rhodamine 6G. Those filters or a well characterized absorber with sharp bands like benzene vapor can be used for the same purpose. The wavelength calibration accuracy obtained with this method is better than 0.1 nm, and requires no modification in the geometry of the spectrofluorometer sample compartment.