Nonlinear Fluorescence Research Articles

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging.

Two-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580-1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two-photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two-photon and the one-photon emission spectra. However, a blue-shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non-linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

Multi-photon Absorption Induced Fluorescence of a Novel Compound

The linear and nonlinear fluorescence spectra of5-(9-anthryl)-3-(4-nitrophenyl)-1-phenyl-2-pyrazoline (ANPP) have beenstudied in different polarity solvents. The linear fluorescence spectrum ofANPP has two emission bands originating from the anthryl and pyrazolinemoieties of ANPP on excitation at 355 nm. The nonlineartwo-photon and three-photon fluorescence from these two moieties areobserved simultaneously when ANPP is excited at 1064 nm due to the proximityof the absorption bands of these two moieties to λ/3 and λ/2 of theexciting wavelength. The similar spectrum structure indicates that thenonlinear and linear fluorescence originates from the same relaxationprocess.

An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules.

Two procedures based on the weighted least-squares (LS) and the maximum likelihood estimation (MLE) method to confidently analyze single-molecule (SM) fluorescence decays with a total number (N) of 2,500-60,000 counts have been elucidated and experimentally compared by analyzing measured bulk and SM decays. The key observation of this comparison is that the LS systematically underestimates the fluorescence lifetimes by approximately 5%, for the range of 1,000-20,000 events, whereas the MLE method gives stable results over the whole intensity range, even at counts N less than 1,000, where the LS analysis delivers unreasonable values. This difference can be attributed to the different statistics approaches and results from improper weighting of the LS method. As expected from theory, the results of both methods become equivalent above a certain threshold of N detected photons per decay, which is here experimentally determined to be approximately 20,000. In contrast to the bulk lifetime distributions, the SM fluorescence lifetime distributions exhibit standard deviations that are sizably larger than the statistically expected values. This comparison proves the strong influence of the inhomogenuous microenvironment on the photophysical behavior of single molecules embedded in a 10-30-nm thin polymer layer.

Time-resolved nonlinear laser-induced fluorescence technique for a combined lifetime and branching-fraction measurement

The single-shot time-resolved laser-induced fluorescence (LIF) technique is well established for measuring radiative lifetimes of atomic states. On top of that, we show that the branching fraction of the pumped transition can be determined and hence also the absolute transition probability, provided that the LIF signal has been sufficiently saturated by the laser pulse. This additional information is solely deduced from the temporal nonlinear behavior of the LIF signal during laser pumping. The alternative procedure is based on the fact that atoms that decay spontaneously into the initial state can be re-excited during the same laser pulse, whereas atoms that decay into third levels cannot. First measurements of this kind have been performed on singly ionized iron. Lifetimes, branching fractions, and resulting transition probabilities are presented and compared with literature data.

Rapid simultaneous determination of four anthracene derivatives using a single non-linear variable-angle synchronous fluorescence spectrum.

A rapid, simple and inexpensive spectrofluorimetric method has been developed for the simultaneous identification and quantification of anthracene (ANT), 9,10-dimethylanthracene (DIM), 2-aminoanthracene (AMI) and dibenz[ah]anthracene (DIB). A well-resolved spectrum for the mixture of these four compounds is obtained based on a single non-linear variable-angle synchronous scanning. The linear concentration ranges are 10-1,000, 5-500, 50-1,000 and 10-200 ng mL(-1) for ANT, DIM, AMI and DIB, respectively, at lambdaex/lambdaem = 358/380, 399/408, 414/465 and 298/394 nm, respectively. The analyses are performed in cyclohexane. Recoveries of 90.0-111.0% in synthetic mixtures are obtained. The detection limits are 2.0 ng mL(-1) for DIM, 2.7 ng mL(-1) for ANT, 15.8 ng mL(-1) for AMI and 4.2 ng mL(-1) for DIB. The method has also been applied to several real water samples with satisfactory results.

Organic nanocrystals embedded in sol–gel glasses for optical applications

We report a simple and generic preparation of stable organic nanocrystals embedded in sol–gel glasses. Nanocrystallization is obtained by instantaneous nucleation followed by controlled growth of the nuclei. In this process, the gel viscosity lowers the growth rate (slow solute diffusion) and inhibits coalescence of the crystals, and pores in the gel act as nanoscale growth reactors. The process is based on control of the nucleation and growth kinetics of the organic phase. For bulk samples, transmission electron microscopy revealed monodisperse particles between 20 and 80 nm in diameter but lower particle diameters are certainly possible due to the nanometer size of pores in dense gel matrix. The generality of this process was demonstrated using various organic molecules selected for their luminescent and optical limiting properties. We have extended this method to the preparation of stable organic nanocrystals embedded in sol–gel thin films using the spin-coating method. These films, which are around 1 mm thick are transparent between 0.3 and 1.5 mm and contain organic crystals characterized by electron and confocal microscopies (100–500 nm in diameter). The targeted properties and applications are electroluminescence, microcavity lasers or nanocrystal spectroscopy. These materials not only combine the optical properties (nonlinear response and fluorescence) of organic molecules with those of inorganic compounds (high stability, wide transparency range) but also combine the advantages of crystals (size effects, photostability) with those of amorphous phases (convenient processing and shaping).

Far-field fluorescence microscopy with repetitive excitation

We introduce the concept of repeatedly exciting an excited state of a photostable fluorescent entity to generate a nonlinear fluorescence signal which is solely based on the linear susceptibility of the molecule. The excitation cycle between the fluorescent state \(\) and another state prolongs the average lifetime of \(\), with emphasis on those molecules that are in the center of the focus. The photons emitted by the long-lived molecules in the center are recorded by a temporal filter and constitute fluorescence that depends nonlinearly on the excitation intensity. Theoretical analysis reveals that this concept can provide three-dimensional imaging and improve the spatial resolution in far-field fluorescence microscopy. We show that despite the presence of diffraction the effective focal waist can in principle be narrowed down to the molecular scale at the expense of signal.

Sensitization mechanisms from excited-singlet state of pyrromethene dye to a radical-generating reagent in a poly(methylmethacrylate) film

The sensitization mechanisms of a pyrromethene dye with a radical-generating reagent, 3,5,3′,5′-tetramethylpyrromethene-BF2 (BH) with 3,3′,4,4′-tetrakis(t-butyldioxycarbonyl)benzophenone (BP), in a poly- (methylmethacrylate) (PMMA) film were investigated by laser flash phoptolysis using a total reflection cell and single photon counting. From the laser flash photolysis, strong fluorescence was detected though no transient absorption was detected. The fluorescence intensity was significantly decreased with increasing concentration of BP, apparently exhibiting Perrin-type static quenching at a quenching radius, Rf = 26 A. From the examination of decay profile using single photon counting, logarithmic plots of fluorescence decay in a PMMA film afforded a nonlinear, convex reduction, corresponding to a streched exponential decay, while the logarithmic plots in acetonitrile showed a linear relationship. With increasing concentration of BH, the fluorescence maximum was shifted to red, and the intensity of fluorescence was significantly reduced. The red shift of fluorescence, the nonlinear fluorescence logarithmic decay and the large reduction in fluorescence indicate a dispersive photoexcited state and a relaxation of excitation energy hopping across an array of sites with Gaussian energy distribution. Moreover, after incorporating BP, the convex logarithmic plots became more steep, and the fluorescence maximum was also shifted to red, exhibiting a nonstatic quenching process competitive to the excitation energy hopping. Thus the sensitization of photoinitiator system containing BH and BP, whose contents were almost same as that in the commercial products, was due to a static quenching process from dispersive singlet excited BH to BP ground state, and the nonstatic quenching process competitive to the excitation energy hopping was minimal. Copyright © 1999 John Wiley & Sons, Ltd.

Sensitization mechanisms from excited‐singlet state of pyrromethene dye to a radical‐generating reagent in a poly(methylmethacrylate) film

The sensitization mechanisms of a pyrromethene dye with a radical-generating reagent, 3,5,3′,5′-tetramethylpyrromethene-BF2 (BH) with 3,3′,4,4′-tetrakis(t-butyldioxycarbonyl)benzophenone (BP), in a poly- (methylmethacrylate) (PMMA) film were investigated by laser flash phoptolysis using a total reflection cell and single photon counting. From the laser flash photolysis, strong fluorescence was detected though no transient absorption was detected. The fluorescence intensity was significantly decreased with increasing concentration of BP, apparently exhibiting Perrin-type static quenching at a quenching radius, Rf = 26 Å. From the examination of decay profile using single photon counting, logarithmic plots of fluorescence decay in a PMMA film afforded a nonlinear, convex reduction, corresponding to a streched exponential decay, while the logarithmic plots in acetonitrile showed a linear relationship. With increasing concentration of BH, the fluorescence maximum was shifted to red, and the intensity of fluorescence was significantly reduced. The red shift of fluorescence, the nonlinear fluorescence logarithmic decay and the large reduction in fluorescence indicate a dispersive photoexcited state and a relaxation of excitation energy hopping across an array of sites with Gaussian energy distribution. Moreover, after incorporating BP, the convex logarithmic plots became more steep, and the fluorescence maximum was also shifted to red, exhibiting a nonstatic quenching process competitive to the excitation energy hopping. Thus the sensitization of photoinitiator system containing BH and BP, whose contents were almost same as that in the commercial products, was due to a static quenching process from dispersive singlet excited BH to BP ground state, and the nonstatic quenching process competitive to the excitation energy hopping was minimal. Copyright © 1999 John Wiley & Sons, Ltd.

Nonlinear Stern-Volmer Fluorescence Quenching of Pyrene by C60/70

The Stern-Volmer plot of fluorescence quenching of pyrene by C60 (or C70) is upward, and non linearly proportional to the fullerene concentration. Electronic absorption measurements show that pyrene and C60 do not form static complexes under the experimental condition. a novel “inner filter effect” model is proposed to rationalize the observed non linear upward Stern Volmer behavior. At a given C60 concentration, increasing either solvent polarity or temperature (T > 280 K) causes diminishment in the I0/I ratio. the data strongly suggest that even at concentrations far below saturation, fullerenes form aggregates in organic solvents.

Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy

We investigate a novel concept to efficiently generate multiphoton induced fluorescence from organic molecules. The concept is based on frustrating the energy transfer between a fluorescent donor and one or more acceptors in conjugated molecules. The nonlinearity is not based on higher order molecular susceptibilities but entirely on their linear properties. Therefore, in contrast to nonresonant multiphoton absorption, this method does not require high local intensities. Likewise, the production of visible fluorescence does not require an infrared excitation wavelength. Hence, when applied to scanning microscopy this property is predicted to increase spatial resolution. Instead of the ∼10 GW/cm2 required in non-resonant multiphoton excitation, focal intensities ∼10 MW/cm2 are expected to produce an equally strong nonlinear signal. The predicted resolution is up to 30% greater than that of an ideal confocal microscope operating at the same fluorescence wavelength. The resolution improvement over non-resonant two-photon absorption microscopes is about two-fold in all directions.

Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy

AbstractWe investigate a novel concept to efficiently generate multiphoton induced fluorescence from organic molecules. The concept is based on frustrating the energy transfer between a fluorescent donor and one or more acceptors in conjugated molecules. The nonlinearity is not based on higher order molecular susceptibilities but entirely on their linear properties. Therefore, in contrast to nonresonant multiphoton absorption, this method does not require high local intensities. Likewise, the production of visible fluorescence does not require an infrared excitation wavelength. Hence, when applied to scanning microscopy this property is predicted to increase spatial resolution. Instead of the ∼10 GW/cm2 required in non‐resonant multiphoton excitation, focal intensities ∼10 MW/cm2 are expected to produce an equally strong nonlinear signal. The predicted resolution is up to 30% greater than that of an ideal confocal microscope operating at the same fluorescence wavelength. The resolution improvement over non‐resonant two‐photon absorption microscopes is about two‐fold in all directions.

`Dressed' atom radiation processes which are first order in the weak field

Nonlinear processes of light scattering on a two-level system near resonance are considered. The problem is reduced to the emission and absorption of a coupled system, formed by a strong resonant field and a two-level system, having a non-factorizing wave function. Various regimes of emission and absorption of the coupled system are determined by the parameter α=|2 d E / ℏΔ|, where E is the amplitude of the strong resonant field, d is the electric dipole moment of the transition 1–2 of the unperturbed atom, Δ is the detuning off resonance, Δ= E 2− E 1− ℏω, with E 1,2 being the energies of, respectively, lower and upper atomic levels, and ω the carrier frequency of the resonant field. Two limiting cases of switching on the interaction between the atom and field are considered, those of the adiabatic and the sudden switching on; these two cases allow simple analytical solutions and lead to essentially differing physical results, either for the probabilities of the processes of the first order with respect to the weak field, or for their coherent properties. Note that at α≪1 the conventional perturbation technique is applicable. This means that for weak fields we can use perturbation method because the corresponding α′≪1. The nonlinear resonance fluorescence and the amplification of weak radiation field by the coupled system at optical frequencies are considered. The coherent properties of the emission of the coupled system are studied which is important for the problems of propagation in gaseous media, coherence and quantum computation.

First-derivative non-linear variable-angle synchronous fluorescence spectroscopy for the simultaneous determination of salicylamide, salsalate and naproxen in serum and urine

Derivative variable-angle synchronous fluorescence spectrometry is developed to improve the selectivity of fluorescence measurements without loss of sensitivity. The first derivative non-linear variable-angle synchronous scanning permits the rapid simultaneous determination of salicylamide, salsalate and naproxen in a mixture from a single spectrum based on a single scan. The method is based on the intrinsic fluorescence of salicylamide, salsalate and naproxen in chloroform. The analyses are performed in the organic phase, using an alkaline medium provided by the addition of 0.40 M pyrrolidine chloroformic solution. The range of application is 0.100–1.000 μg/ml for all of them. A systematic examination of the experimental data by applying an exhaustive statistical analysis is reported. The method is applied in urine and serum samples spiked with all the drugs. Serum and urine samples are extracted into chloroform–1% acetic acid solution, using in the aqueous medium a pH of 4.8 obtained by the addition of acetate buffer solution, and a basic medium is provided with pyrrolidine prior to instrumental measurement. The simultaneous determination of salicylamide, salsalate and naproxen in human serum and urine samples is performed with this technique in the extracted chloroform phase, without necessity of any re-extraction step. The validity, applicability and simplicity of the method are demonstrated.

Simultaneous determination of atenolol, propranolol, dipyridamole and amiloride by means of non-linear variable-angle synchronous fluorescence spectrometry

First derivative non-linear variable-angle synchronous fluorescence spectrometry has been developed to improve the selectivity of fluorescence measurements without loss of sensitivity. It allows the rapid simultaneous determination of different substances in a mixture from a single spectrum based on a single scan. This method was applied for the simultaneous determination of atenolol (ATE), propranolol (PRO), amiloride (AMI) and dipyridamole (DIP) at concentrations between 10–400, 6–200, 5.6–280 and 5–100 ng ml −1, respectively, by means of absolute values of first derivative of non-linear variable-angle synchronous scan at λ ex/ λ em=228.8/300, 287.2/340, 366.4/412.8 and 288/487.2 nm for ATE, PRO, AMI and DIP, respectively. In order to obtain maximum sensitivity and an adequate selectivity, factors affecting fluorescence intensity were studied. As a result, the analyses were performed in an ethanol–water (70%(v/v)) medium at a pH 7.5, adjusted by using trishydroxymethyl amino methane (0.08 M) as a buffer solution. Analytical parameters of the proposed method were calculated according to the error propagation theory. The sensitivity, repeatability, reproducibility and limit of detection achieved with the proposed method are adequate for the determination of these doping substances.

Optical Monitoring of Living Brain Tissue

Optical methods can usefully augment classical techniques such as electrical recording for investigating cellular and network physiology. Here, recent developments in optical methods--including confocal and nonlinear fluorescence imaging, semiconductor array imaging, and improved fluorescent indicators of ion concentration and transmembrane electrical potential--are briefly reviewed, and examples are offered that may suggest potential applications to experimental studies in alcoholism research.

Time-resolved nonlinear fluorescence spectroscopy using femtosecond multiphoton excitation and single-photon timing detection

We have developed a time-correlated single-photon timing nonlinear fluorometer for recording the fluorescence decay times and rotational correlation times of molecular probes using 120 fs regeneratively amplified Ti:Sapphire laser excitation via simultaneous non-resonant absorption of two or more near infrared photons. A microchannel plate photomultiplier giving 70 ps impulse response is used for detection. Studies on 1,6-diphenylhexatriene, rhodamine 6G and p-terphenyl in propylene glycol demonstrate two- and three-photon induced fluorescence characteristics. The radiative properties for one- and multiphoton excitation were found to be identical. However, the time-zero anisotropy observed for multiphoton excitation was larger than for one-photon excitation, indicating an increased degree of orientation of excited molecules after multiphoton absorption. The results reveal the potential of multiphoton-induced fluorescence anisotropy in the study of the structure and dynamics of microheterogeneous systems (i.e. biomembranes, porous matrices etc) by selecting the excitation wavelength and class of probe molecule.

Application of Multiphoton Imaging to Study of the Vasculature.

Nonlinear or multiphoton fluorescence microscopy utilizes the simultaneous absorption of two (or three) longer wavelength photons to excite fluorophores (Denk et.al., 1990). For example, UV absorbing fluorophores such as DAPI or lndo-1 are excited using 700 nm near infrared light rather than 350 nm UV excitation. This relatively new form of laser scanning fluorescence microscopy has excellent optical sectioning capabilities in thick, highly scattering tissues. The high degree of intrinsic optical sectioning arises from the spatial nature of the excitation dependence, rather than from a confocal aperture or deconvolution algorithm. The fluorescence arising from any point in the specimen depends on the second or third power of the intensity (i.e. two and three photon excitation, respectively). This squaring (or cubing) of the illumination point spread function effectively confines the excitation to a tenth of a femtoliter optical volume when using a high NA lens. 680 to 1100 nm mode-locked lasers producing pulses of 100 femtosecond duration at a repetition rate of 80 Mhz are used to efficiently produce fluorescence excitation at average powers that are usually in the 0.5 to 10 mW range at the sample.

Multivariate nonlinear modelling of fluorescence data by neural network with hidden node pruning algorithm

A hidden node pruning algorithm (HNPA) has been proposed as a method of configuration optimization and training in multilayer feedforward network. By this approach, a network initially bearing excessive hidden nodes is trained to sufficient precision and is pruned to the optimal size. Upon pruning, significant hidden nodes are determined by singular value decomposition (SVD) of output matrix of hidden layer and are retained. Weights and biases are preset intentionally in the pruned system, then training continues. The method has been tested with simulated nonlinear data and then applied to the modelling of a nonlinear fluorescence data of a real multicomponent analytical system, and satisfactory quantitative results have been achieved.

Rapid resolution of five polynuclear aromatic compounds in a mixture by derivative non-linear variable-angle synchronous fluorescence spectrometry

A combination of non-linear variable-angle synchronous fluorescence spectrometry with the derivative technique has been developed. A rapid simultaneous identification and quantitative determination of acenaphthene, carbazole, anthracene, 9,10-dimethylanthracene and perylene were achieved from a single spectrum by non-linear variable-angle synchronous fluorescence and combined derivative non-linear variable-angle synchronous fluorescence. The latter approach offered a further improvement in spectral resolution and analytical sensitivity. The usefulness of the proposed method was confirmed by adding known amounts of these five polynuclear aromatic compounds to the extract of waste water samples.