Two-photon absorption-induced optical properties of a new lasing dye in two solvents

Pumped by an infrared lasing and its frequency-doubling source, the fluorescence and superradiance emission properties of a new lasing dye trans-4-[4^′-(N,N-diethylamino)styryl]-N-methyl pyridinium methyl sulfate (abbreviated DEASPS), have been studied in benzyl alcohol and chloroform. The two-photon absorption (TPA) and emission properties of DEASPSare influenced by the solvents used. The emission wavelength of the dye in benzyl alcohol is redshifted relative to that in chloroform. The lifetime of two-photon absorption-induced fluorescence is about 529 ps in benzyl alcohol, whereas it is 340 ps in chloroform. Correspondingly, the one-photon-excited fluorescence lifetimes in the two solvents are also given. The upconversion efficiency of DEASPS in chloroform is higher than in benzyl alcohol. Finally, the effective molecular two-photon absorption cross-sections were measured by the nonlinear transmittance method. It is found that the optical limiting effect of the dye in benzyl alcohol is stronger than that in chloroform. Received: 3 May 2001 / Revised version: 6 July 2001 / Published online: 19 September 2001     

Evanescent-field-induced two-photon fluorescence: excitation of macroscopic areas of planar waveguides

Two-photon excitation (TPE) of fluorescence is a powerful tool for separating a faintly emitted fluorescence signal from background excitation noise. Until now TPE has only been accomplished for very small excitation areas of a few square micrometre dimensions since they are readily available in the focal zone of high-power lasers. In this paper we demonstrate, to our knowledge for the first time, two-photon excited fluorescence with planar thin-film waveguide structures of macroscopic excitation areas of the order of square millimetres to square centimetres. Based upon our results, it can be envisaged that the new combination of planar waveguide technology with TPE will become a powerful tool for macroscopic surface-interaction studies. It can also open the way for further improving the sensitivity of biosensing platforms like genomic and proteomic microarrays based upon planar waveguides. Received: 7 November 2001 / Published online: 23 November 2001     

Two-Photon Activated Two-Photon Fluorescence and Binding of Azidocoumarin in a Gelatin Matrix

We study the creation of fluorescence patterns inside a gelatin gel by way of two-photon photoactivation of 7-azido-4-trifluoromethyl-1,2-benzopyrone (azidocomarin 151) contained in the gel matrix. As ultrafast light pulses are focused into the gel, onset of two-photon fluorescence, highly nonlinear in the applied optical power, is observed as azidocoumarin is converted into a fluorescent dye that binds to the gelatin. We fit the time dependence of the fluorescence to a model that incorporates the competition between coumarin photoactivation and photobleaching as well as the gradual degradation of the gel when it is exposed to the high intensity laser light. The model predicts that the initial rate of fluorescence onset should scale as the P (4), where P is laser power, while the signal at long exposure time should scale as P (3/2). The observed exponents are 4.18 and 1.34, respectively. The model allows us to estimate the cross section and quantum yield of two-photon induced photobleaching of azidocoumarin 151. The numerous technical uses of gelatin and the collagen from which it derives in areas ranging from photography to tissue engineering provide possible applications for the techniques described in this paper.     

Greater signal, increased depth, and less photobleaching in two-photon microscopy with 10 fs pulses

The fundamental advantages to using ultrafast (?100 fs) laser pulses in two-photon microscopy for biomedical imaging are seldom realized due to chromatic dispersion introduced by the required high numerical aperture microscope objective. Dispersion is eliminated here by using the multiphoton intrapulse interference phase scan (MIIPS) method on pulses with a bandwidth greater than 100 nm full width at half maximum. Higher fluorescence intensity, deeper sample penetration, and improved signal-to-noise ratio are demonstrated quantitatively and qualitatively. Due to the higher signal intensity obtained after MIIPS compensation, lower laser power is required, which decreases photobleaching. The observed advantages are not realized if group delay dispersion is compensated for while higher-order dispersion is not. By using a pulse shaper and taking advantage of the broad spectrum of the ultrafast laser, selective excitation of different cell organelles is achieved due to the difference in nonlinear optical susceptibility of different chromophores without requiring an emission filter wheel. Experiments on biological specimens, such as HeLa cells and mouse kidney tissue samples, illustrate the advantages to using sub-10 fs pulses with MIIPS compensation in the field of two-photon microscopy for biomedical imaging.     

Independence of Maximum Single Molecule Fluorescence Count Rate on the Temporal and Spectral Laser Pulse Width in Two-Photon FCS

We investigate the fluorescence emission characteristics of standard dye tetramethylrhodamine (TMR) in two-photon fluorescence correlation spectroscopy for different temporal and spectral properties of the femtosecond excitation pulses. After determining the second-order dispersion of our setup, including the microscope objective, a pulse stretcher was employed to control the temporal width at the location of the specimen. As expected, the fluorescence per molecule and therefore the signal-to-noise ratio of an FCS-measurement can be improved at constant average excitation power by altering either the temporal or spectral width of the excitation pulses. We found however, that the maximum achievable molecular brightness is largely independent of the temporal and spectral width in the regime analyzed. This observation confirms the current working hypothesis for two-photon fluorescence correlation spectroscopy that bleaching and saturation, and thus, the inherent properties of the dye system, are the dominant effects limiting the quality of measurements. As a practical consequence, elaborate optimization of temporal and spectral laser pulse width, e.g. by introducing pulse stretchers in the beam path, is less critical than previously expected.     

Photobleaching on Photonic Crystal Enhanced Fluorescence Surfaces

The effect of resonant fluorescent enhancement from a photonic crystal surface upon the fluorescent photobleaching rate of Cyanine-5 labeled protein has been investigated. We show that the enhanced excitation mechanism for photonic crystal enhanced fluorescence, in which the device surface resonantly couples light from an excitation laser, accelerates photobleaching in proportion to the coupling efficiency of the laser to the photonic crystal. We also show that the enhanced extraction mechanism, in which the photonic crystal directs emitted photons approximately normal to the surface, does not play a role in the rate of photobleaching. We show that the photobleaching rate of dye molecules on the photonic crystal surface is accelerated by 30x compared to an ordinary glass surface, but substantial signal gain is still evident, even after extended periods of continuous illumination at the resonant condition.     

Photobleaching, Mobility, and Compartmentalisation: Inferences in Fluorescence Correlation Spectroscopy

In living cells the transport and diffusion of molecules is constrained by compartments of various sizes. This paper is an attempt to show that the size of these compartments can in principle be estimated experimentally from Fluorescence Correlation Spectroscopy (FCS) combined with the measurement of the photobleaching rate. In this work, confocal fluorescence microscopy experiments have been carried out on giant unilamellar vesicles, a system that mimics cellular compartmentalisation. We have developed numerical and analytical models to describe the fluorescence decay due to photobleaching in this geometry, which has enabled us to point out two regimes depending on the value of the parameter P(B) = sigma(B)P/D (where sigma(B) is the photobleaching cross section of the dye, D its diffusion constant, and P the laser power (in photon/s)). In particular, when P(B) < 1 (i.e. in the fast diffusion regime), the photobleaching rate is independent of the diffusion constant and scales like sigma(B)P/R2, in agreement with the experimental results. On the other hand, the standard diffusion models used to analyse the FCS data do not take into account the effects of the fluorescence decay on the autocorrelation curve. We show here how to correct the raw data for these drawbacks.     

Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy

In this work we investigated the relative merits of conventional single-photon confocal laser scanning fluorescence microscopy (CLSM) and two-photon laser scanning fluorescence microscopy (2p-LSM) for the study of mitochondria in living neurons. Dorsal root ganglion neurons were loaded with the mitochondrion-specific fluorescent dye JC-1, the ratio between red (J-aggregates) and green (monomer) fluorescence of which reflects mitochondrial membrane potential. Cells were illuminated at 488 nm for single-photon excitation or at 870 nm for two-photon excitation. In both modalities we found that mitochondria showed: (i) similar appearance; (ii) similar fluorescence ratio values over both whole cell bodies and individual mitochondria; and (iii) similar responses to mitochondrial uncoupler, which dropped the ratio values by 50%. However, 2p-LSM exhibited several advantages over CLSM: (i) better signal/noise ratio in the green emission channel; (ii) less phototoxicity upon repetitive scanning in the focal plane; and (iii) no significant loss of image quality upon repetitive scans in the z direction. We conclude that, while both techniques enable visualisation of individual mitochondria in living cells, 2p-LSM has significant advantages for physiological work requiring time-lapse experiments or four-dimensional reconstructions of mitochondria.     

Electrically switchable reflection holograms formed using two-photon photopolymerization

Two-photon holographic photopolymerization was used to form switchable Bragg gratings composed of layers of phase-separated liquid-crystal (LC) domains interspersed with cured, crosslinked polymer. These holographic polymer-dispersed liquid crystals form a periodic structure which diffracts red light due to nanostructured planes ∼250 nm in spacing. These structures were formed by interfering two 90-fs pulses coherently upon a reactive syrup consisting of acrylate monomer, liquid crystal, and a two-photon dye. The large two-photon cross-section allows excitation of the two-photon dye that results in electron transfer between this dye and the monomer. Diffraction efficiencies of approximately 10% were obtained, which can be modulated using an electric field applied across the film. Switching speeds below 1 ms were observed due in part to the small size of the LC domains. Received: 10 April 2001 / Accepted: 1 July 2001 / Published online: 2 October 2001     

Fluorescence Study of DNA–Dye Complexes Using One-Photon and Two-Photon Picosecond Excitation

Preliminary results of investigation of one-photon- and two-photon-induced fluorescence of acridine orange (AO), epirubicin (ER), hypericin (HYP), and ethidium bromide (EB) in complexes with DNA are presented. A spectrofluorometer based on a picosecond Nd:YAG laser was used for investigations of two-photon (1064-nm, 1-mJ, 40-ps) and one-photon (532- and 355-nm) dye excitation. The spectra of two-photon-induced fluorescence of dyes and their complexes with DNA as well as the kinetics of dyes' fluorescence intensification during their interactions with DNA in dependence on the biomacromolecule concentration were obtained. The intensities of AO, HYP, and EB fluorescence were increased 2.4, 3.2, and 8 times, respectively, after binding with DNA at two-photon excitation, while at one-photon excitation the corresponding values were 2.5, 3.7, and 10 times. The difference in fluorescence enhancement during DNA–dye complex formation at linear and nonlinear excitation may possibly be associated with the fact that the cross sections of one-photon and two-photon absorption, in general, change unequally during the binding of dyes to organic molecules and bathocromic shift of the electronic transitions. It was shown that the peak of AO fluorescence shifted to a longer wavelength on 10 nm after two-photon excitation at 1064 nm in comparison with one-photon excitation at 532 nm.     

Spectroscopic and lasing properties of Xanthene dyes encapsulated in silica and polymeric matrices

The photo-physical properties of Xanthene dyes: Basic Rhodamine Yellow (BRY), Rhodamine590perchlorate (R590p), SulforhodamineB (SRB) doped in tetramethylorthosilicate (TMOS) and poly-methylmethacrylate (PMMA) are observed. The various parameters viz. full-width at half-maxima (FWHM), peak emission wavelength, quantum yield and excited state lifetime at different concentrations ranging from ∼0.05 to ∼1 mM of the dye under excitation by Copper Vapor Laser (CVL) of high repetition rate (∼5.6 kHz) of are investigated. In order to identify photostability in dyes, normalized photostability has been studied and found that silica gel samples containing dye are more stable than that of polymeric samples. This has been further understood in terms of number density of unbleached dye molecules that infers that photobleaching of dye molecules is not prominent at higher concentrations in glassy solid matrices. Pump intensity dependent optical gain of the samples has also been reported and efforts have been made to study the efficiency of solid-state laser samples in a cavity for the performance of the dye laser.     

Two-photon excitation of Na atoms in a flame by broad-band laser irradiation

With a flashlamp-pumped tunable dye laser the 3S→3D, 3S→4D and 3S→5S two-photon transitions of sodium are excited and the resulting fluorescence radiation at various atomic transitions is detected. The sodium is nebulized into a stoichiometric H₂-O₂-Ar flame at atmospheric pressure and at a temperature of 1800 K. Collisional population exchange between several of the higher Na-levels is found to occur. Saturation and saturation broadening of two-photon transitions are observed. Formulas for two- photon excitation rates in the case of broad-band excitation are derived and show substantial agreement with experiment.     

Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation

We examined the steady-state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-photon (OPE) and two-photon (TPE) excitation. Hoechst 33342 was found to display a large cross section for two-photon excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for OPE- and TPE. The anisotropy decay measurements of Hoechst 33342 in ethanol revealed the same correlation times for TPE as observed for OPE. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for TPE than for OPE. The anisotropy spectra of Hoechst 33342 were examined in glycerol at ?20°C, revealing limiting values close to the theoretical limits for OPE (0.4) and TPE (0.57). The steady-state anisotropy for OPE decreases in the shorter-wavelength region (R6G dye laser, 280–315 nm), but the two-photon anisotropy for 560 to 630-nm excitation remains as high as in the long-wavelength region (690–770 nm). This result suggests that one-photon absorption is due to two electronic, but only one transition contributes to the two-photon absorption over the wavelength range from 580 to 770 nm. Our demonstration of these favorable two-photon properties for Hoechst 33342, and the high photostability of the dye reported by other laboratories, suggests that this dye will be valuable for time-resolved studies of DNA with TPE and for two-photon fluorescence microscopy.     

Arrangement of a 4Pi microscope for reducing the confocal detection volume with two-photon excitation

The main advantage of two-photon fluorescence confocal microscopy is the low absorption obtained with live tissues at the wavelengths of operation. However, the resolution of two-photon fluorescence confocal microscopes is lower than in the case of one-photon excitation. The 4Pi microscope type C working in two-photon regime, in which the excitation beams are coherently superimposed and, simultaneously, the emitted beams are also coherently added, has shown to be a good solution for increasing the resolution along the optical axis and for reducing the amplitude of the side lobes of the point spread function. However, the resolution in the transverse plane is poorer than in the case of one-photon excitation due to the larger wavelength involved in the two-photon fluorescence process. In this paper we show that a particular arrangement of the 4Pi microscope, referenced as 4Pi′ microscope, is a solution for obtaining a lateral resolution in the two-photon regime similar or even better to that obtained with 4Pi microscopes working in the one-photon excitation regime.     

Two-color two-photon excitation of indole using a femtosecond laser regenerative amplifier

The fluorescence emission from indole resulting from two-color two-photon (2C2P) excitation with 400 and 800 nm wavelengths is observed, using the second harmonic and fundamental wavelength of a 800 nm 40 fs pulsed Ti:Sapphire femtosecond (fs) regenerative amplifier operating at a repetition rate of 1 kHz. By delaying one fs laser pulse relative to the other, the cross correlation of fluorescence is observed, which indicates the generation of 2C2P fluorescence signal in the experiment. The strongest 2C2P fluorescence emission characterized by the peak of cross correlation curve suggests optimal temporal overlap of the two fs laser pulses. The 2C2P fluorescence signal is linearly dependent on the total excitation intensity. The fluorescence signals with 400 nm and 800 nm irradiation alone are also demonstrated and discussed in this paper.     

Absorption and Emission Properties of a New Two-photon Absorption Dye

The emission properties of a newly synthesized organic two-photon absorption dye, trans-4-[p-(N-ethyl-N-ethylamino)-styryl]-N-methyl-pyridinium tetraphenylborate, have been investigated. When pumped by infrared pulses from a picosecond Nd∶YAG laser, the dye exhibits intense upconverted fluorescence and strong superradiance properties. For comparison, the one-photon induced fluorescence and superradiance are also measured. The one- and two-photon excited fluorescent lifetimes are 86 and 64 ps, respectively. The maximum efficiency of the dye is measured to be 1.97% and the upconversion efficiencies at different pump wavelengths have also been investigated by the optical parametric amplifier.     

Absorption and Emission Properties of a New Two-photon Absorption Dye

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Temporal and spectral properties of picosecond two-photon pumped cavity lasing of an organic dye HEASPS

The operation of cavity lasing of a two-photon absorption (TPA) organic dye, trans-4-[p-(N-hydroxyethyl-N-ethylamino)styryl]-N-methylpyridinium p-toluene sulfonate (abbreviated as HEASPS), by using a picosecond infrared laser as the pump source, is reported. The lifetimes of TPA fluorescence of this dye in different solvents were measured. Temporal profiles of cavity lasing show obvious oscillations and magnification of the feedback light. By using the difference of re-absorption coefficients at different conditions, we explained the blue shift for cavity lasing and superradiance compared with TPA-induced fluorescence. The long lifetime of fluorescence is helpful to the generation of cavity lasing. Received: 18 June 2001 / Revised version: 5 November 2001 / Published online: 17 January 2002     

Room temperature stable single-photon source

We report on the realization of a stable solid state room temperature source for single photons. It is based on the fluorescence of a single nitrogen-vacancy (NV) color center in a diamond nanocrystal. Antibunching has been observed in the fluorescence light under both continuous and pulsed excitation. Our source delivers 2×10⁴ s^-1 single-photon pulses at an excitation repetition rate of 10 MHz. The number of two-photon pulses is reduced by a factor of five compared to strongly attenuated coherent sources. Received 1st August 2001 and Received in final form 2 October 2001     

Two photon-induced fluorescence intensity and anisotropy decays of diphenylhexatriene in solvents and lipid bilayers

We measured the fluorescence intensity and anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled membranes resulting from simultaneous two-photon excitation of fluorescence. Comparison of these two-photon data with the more usual one-photon measurements revealed that DPH displayed identical intensity decays, anisotropy decays, and order parameters for one- and two-photon excitation. While the anisotropy data are numerically distinct, they can be compared by use of the factor 10/7, which accounts for the two-photon versus one-photon photoselection. The increased time 0 anisotropy of DPH can result in increased resolution of complex anisotropy decays. Global analysis of the one- and two-photon data reveals consistency with a single apparent angle between the absorption and the emission oscillators. The global anisotropy analysis also suggests that, except for the photoselection factor, the anisotropy decays are the same for one-and two-photon excitation. This ideal behavior of DPH as a two-photon absorber, and its high two-photon cross section, makes DPH a potential probe for confocal two-photon microscopy and other systems where it is advantageous to use long-wavelength (680- to 760-nm) excitation.