Two-photon Fluorescence Excitation Spectra Research Articles

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

(A)symmetric chromophores based on cyano and fluorine-substituted 2,3-bis(5-arylthiophen-2-yl)quinoxalines: Synthesis, photophysical properties and application prospects

A series of novel V-shaped D-π-A-π-D push-pull chromophores, containing two aminoaryl electron donors (D), 1,5-thiophenylene π-conjugated spacer and a 6-cyano- or 6,7-difluoroquinoxaline electron acceptor (A), was designed, synthesized, and characterized. 6-Cyano derivatives demonstrated red shift of absorption and emission maxima compared with 6,7-difluoro counterparts. The compounds displayed positive emission solvatochromism with increasing solvent polarity. Additionally, emission behavior of all compounds in THF/water mixture and solid state was investigated. Moreover, the two-photon excited fluorescence (2 PE) spectra of carbazolyl-containing chromophores were measured. Replacement of 6,7-difluoroquinoxaline core by 6-cyanoquinoxaline increases two-photon absorption cross section (δ2PA) value by more than 10 times. 6,7-Difluoroquinoxalines demonstrated changes in emission maxima or/and intensity after mechanic stimulation. Electronic-structure calculations with the use of quantum-chemistry methods were performed to have a deeper insight into experimental data. In summary, the obtained results show multifunctional nature of V-shaped 2,3-bis(5-arylthiephen-2-yl)quinoxalines and their potential utility in diverse applications.

Selecting optimal spectral bands for improved detection of autofluorescent biomarkers in multiphoton microscopy.

.Significance: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing.Aim: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers.Approach: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio.Results: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images.Conclusions: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.

Laser-Induced Fluorescence Investigations for Temperature Measurements in a Carbon Dioxide Flow

The objective of the presented work was the investigation of two-photon laser-induced fluorescence excitation spectra of carbon monoxide (CO) molecules (B-X) in a carbon dioxide () flow with an enthalpy of . The flow was generated with an arcjet-driven plasma wind tunnel. For validation, different stoichiometric premixed flat flames and a low-pressure cold-gas cell filled with CO have been examined. For the excitation of the Q-branch of the transition, the laser was tuned from 230.04 to 230.11 nm, with a step width of 0.0005 nm. Simulated spectra were calculated in advance with a simulation tool called NOCO-Spectra and showed good agreement with the experimental data. Finally, the rotational temperature of CO in the flow was evaluated by comparison of the CO-excitation spectra and the calculated spectra using a correlation automated rotational fitting procedure.

Ligand-core NLO-phores: a combined experimental and theoretical approach to the two-photon absorption and two-photon excited emission properties of small-ligated silver nanoclusters.

We report a combined experimental and theoretical study of the two-photon absorption and excited emission properties of monodisperse ligand stabilized Ag11, Ag15 and Ag31 nanoclusters in aqueous solutions. The nanoclusters were synthesized using a cyclic reduction under oxidative conditions and separated by vertical gel electrophoresis. The two-photon absorption cross-sections of these protected noble metal nanoclusters measured within the biologically attractive 750-900 nm window are several orders of magnitude larger than that reported for commercially available standard organic dyes. The two-photon excited fluorescence spectra are also presented for excitation wavelengths within the same excitation spectral window. They exhibit size-tunability. Because the fundamental photophysical mechanisms underlying these multiphoton processes in ligand protected clusters with only a few metal atoms are not fully understood yet, a theoretical model is proposed to identify the key driving elements. Elements that regulate the dipole moments and the nonlinear optical properties are the nanocluster size, its structure and the charge distribution on both the metal core and the bound ligands. We coined this new class of NLO materials as "Ligand-Core" NLO-phores.

Surface recombination and charged exciton in nanocrystal quantum dots on photonic crystals under two-photon excitation.

In this study, the two-photon excited fluorescence spectra from cadmium selenide quantum dots (QDs) on a silicon nitride photonic crystal (PhC) membrane under femtosecond laser irradiation were investigated. These spectra can be fit to a tri-Gaussian function in which one component is negative in amplitude, and in which the Gaussian components with positive amplitude are assigned to exciton emission and charged-exciton emission and that with negative amplitude is assigned to absorption from surface recombination. The photonic crystal enhance the charged-exciton emission and exciton emission and, at the same time, also the absorption from surface recombination. Both the charged-exciton emission and the surface recombination are related to Auger recombination; therefore, the photonic crystal controls both radiative recombination and non-radiative recombination. The asymmetries of the two-photon excited fluorescence spectra are due to not only the location of the resonant guide mode of the PhC slab but also the enhancement of the absorption from surface recombination by PhC.

Excitonic splitting and vibronic coupling in 1,2-diphenoxyethane: Conformation-specific effects in the weak coupling limit

Vibrationally and rotationally resolved electronic spectra of 1,2-diphenoxyethane (C6H5-O-CH2-CH2-O-C6H5, DPOE) are reported for the isolated molecule under jet-cooled conditions. The spectra demonstrate that the two excited surfaces are within a few cm(-1) of one another over significant regions of the torsional potential energy surfaces that modulate the position and orientation of the two aromatic rings with respect to one another. Two-color resonant two-photon ionization (2C-R2PI) and laser-induced fluorescence excitation spectra were recorded in the near-ultraviolet in the region of the close-lying S0-S1 and S0-S2 states (36,400-36,750 cm(-1)). In previous work, double resonance spectroscopy in the ultraviolet and alkyl CH stretch regions of the infrared was used to identify and assign transitions to two conformational isomers differing primarily in the central C-C dihedral angle, a tgt conformation with C2 symmetry and a ttt conformation with C2h symmetry [E. G. Buchanan, E. L. Sibert, and T. S. Zwier, J. Phys. Chem. A 117, 2800 (2013)]. Comparison of 2C-R2PI spectra recorded in the m∕z 214 (all (12)C) and m∕z 215 (one (13)C) mass channels demonstrate the close proximity of the S1 and S2 excited states for both conformations, with an upper bound of 4 cm(-1) between them. High resolution spectra of the origin band of the tgt conformer reveal it to consist of two transitions at 36,422.91 and 36,423.93 cm(-1), with transition dipole moments perpendicular to one another. These are assigned to the S0-S1 and S0-S2 origin transitions with excited states of A and B symmetry, respectively, and an excitonic splitting of only 1.02 cm(-1). The excited state rotational constants and transition dipole coupling model directions prove that the electronic excitation is delocalized over the two rings. The ttt conformer has only one dipole-allowed electronic transition (Ag→Bu) giving rise to a pure b-type band at 36,508.77 cm(-1). Here, the asymmetry induced by a single (13)C atom in one of the rings is sufficient to localize the electronic excitation in one or the other ring. Dispersed fluorescence (DFL) spectra are used to provide assignments for all vibronic structure in the first 200 cm(-1)of both conformers. In the tgt conformer, both "a" and "b" symmetry fundamentals are observed, consistent with extensive vibronic coupling between the two dipole-allowed, nearly degenerate excited states. In the ttt conformer, the lowest frequency vibronic transition located 46 cm(-1) above the Bu origin is assigned to a bu fundamental (labeled R[overline]) built off the dipole-forbidden Ag state origin. The DFL spectrum of the Ag(R[overline](1)) level contains strong transitions to v(")(R[overline]) = 0, 1, and 2, seemingly at odds with vibronic coupling models. Studies of the DFL spectrum of this band as a function of distance from the nozzle reveal that much of the intensity in v(") = 1 arises from collisions of DPOE while in the excited state Ag(vb' = 1) level with He, producing Bu(R[overline] = 1) levels with large collision cross section. The remaining intensity in the fundamental at large x∕D is ascribed to emission from the (13)C isotopomer, for which this emission is dipole-allowed.

High-Pressure Optical Properties and Chemical Stability of Picene

Picene is a polycyclic aromatic hydrocarbon belonging to the class of phenacenes which have been recently found to behave as high-temperature superconductors upon alkali metal doping. The electronic properties of organic crystals can be finely and largely modified by the density changes obtained by the application of an external pressure. In this work, the role of pressure in tuning the optical properties of crystalline picene has been investigated from room conditions up to 15 GPa through the measurement of UV–visible absorption spectra, two-photon excitation profiles, and one- and two-photon excited fluorescence spectra in a diamond anvil cell. The pressure dependence of the optical band gap was determined, and the frequencies of several vibronic bands belonging to electronic transitions from the ground state (S0) to the four lowest-energy excited singlet states (S1 to S4) were determined as a function of pressure. We evidence a very different density dependence of the transition energy of S0 → S1, which undergoes a remarkable red shift of ∼400 cm–1/GPa, and of the transitions from S0 to the higher excited states, which remain constant in the whole investigated range. This is consistent with a S1 state of 1La character in solid picene. The high-pressure chemical stability of solid picene was investigated through visible absorption and Fourier transform infrared spectroscopy (FTIR). A chemical transformation involving the bulk picene crystal occurs above ∼23 GPa, giving rise to a disordered material similar to the amorphous hydrogenated carbon obtained in the pressure-induced reactivity of benzene. The combination of electronic and vibrational data allows us to identify the presence of reaction intermediates at ∼10 GPa, preferentially forming at crystal defects.

Two-Photon Absorption Properties of <i>s</i>-Triazine Derivatives With Near Octupolar Symmetry

Two new three-branched s-triazine derivatives with near-octupolar symmetry have been synthesized. In each of them, two branches have donor groups attaching to their ends, while the other one has none. Their photophysical characteristics, such as UV–vis spectra, fluorescence spectra and two-photon excited fluorescence spectra, are compared with their octuplar analogs in which all the three branches are ended by donor group. The little structural difference results in great decline of single-photon absorptivity and single-photon excited fluorescence, as well as the two-photon excitated fluorescence.

Impact of Electronic Coupling, Symmetry, and Planarization on One- and Two-Photon Properties of Triarylamines with One, Two, or Three Diarylboryl Acceptors

We have performed a study of the one- and two-photon absorption properties of a systematically varied series of triarylamino-compounds with one, two, or three attached diarylborane arms arranged in linear dipolar, bent dipolar, and octupolar geometries. Two-photon fluorescence excitation spectra were measured over a wide spectral range with femtosecond laser pulses. We found that on going from the single-arm to the two- and three-arm systems, the peak in two-photon absorption (2PA) cross-section is suppressed by factors of 3-11 for the lowest excitonic level associated with the electronic coupling of the arms, whereas it is enhanced by factors of 4-8 for the higher excitonic level. These results show that the coupling of arms redistributes the 2PA cross-section between the excitonic levels in a manner that strongly favors the higher-energy excitonic level. The experimental data on one- and two-photon cross-sections, ground- and excited-state transition dipole moments, and permanent dipole moment differences between the ground and the lowest excited states were compared to the results obtained from a simple Frenkel exciton model and from highly correlated quantum-chemical calculations. It has been found that planarization of the structure around the triarylamine moiety leads to a sizable increase in peak 2PA cross-section for the lowest excitonic level of the two-arm system, whereas for the three-arm system, the corresponding peak was weakened and shifted to lower energy. Our studies show the importance of the interarm coupling, number of arms, and structural planarity on both the enhancement and the suppression of two-photon cross-sections in multiarm molecules.

Pressure-Induced Fluorescence of Pyridine

Two-photon excitation profiles and fluorescence spectra have been measured as a function of pressure in a diamond anvil cell up to 15.5 GPa in crystal phases I and II and in the glassy form of pyridine. The fluorescence emission intensity increases by about 6 orders of magnitude in going from the liquid to the crystalline phases at 3 GPa and further increases with pressure. This is explained by an energy inversion of the lowest (1)B(1) (nπ*) and (1)B(2) (ππ*) excited states likely due to the involvement of the lone pair of the N atom in intermolecular CH···N bonds. These interactions characterize the crystal phases and are stabilized by pressure. The glassy form, accordingly, is characterized by a much weaker fluorescence. Excimer emission is also observed. Comparison of the emission of several samples with different compression and annealing histories, the lack of reversibility in the excimer emission with decompression, and the larger relative intensity of the excimer band in the glassy form suggest that excimer formation occurs at crystal defects. This results support the conclusions of a previous investigation proposing that pressure-induced reactivity of pyridine is limited to crystal defects and agrees with the present knowledge of the solid-state chemistry of aromatic crystals.

Two-photon fluorescence excitation spectroscopy by pulse shaping ultrabroad-bandwidth femtosecond laser pulses

A fast and automated approach to measuring two-photon fluorescence excitation (TPE) spectra of fluorophores with high resolution (~2 nm) by pulse shaping ultrabroad-bandwidth femtosecond laser pulses is demonstrated. Selective excitation in the range of 675-990 nm was achieved by imposing a series of specially designed phase and amplitude masks on the excitation pulses using a pulse shaper. The method eliminates the need for laser tuning and is, thus, suitable for non-laser-expert use. The TPE spectrum of Fluorescein was compared with independent measurements and the spectra of the pH-sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in acidic and basic environments were measured for the first time using this approach.

Crystalline Indole at High Pressure: Chemical Stability, Electronic, and Vibrational Properties

Vibrational and electronic spectra of crystalline indole were measured up to 25.5 GPa at room temperature in a diamond anvil cell. In particular, Fourier transform infrared (FTIR) spectra in the mid-infrared region and two-photon excitation profiles and fluorescence spectra in the region of the HOMO-LUMO transitions were obtained. The analysis of the FTIR spectra revealed a large red-shift of the N-H stretching mode with increasing pressure, indicating the strengthening of the H-bond between the NH group and the pi electron density of nearest neighbor molecules. The frequencies of four vibronic bands belonging to the (1)L(a) and (1)L(b) systems were obtained as a function of pressure. Comparison with literature data shows that the crystal acts as a highly polar environment with regard to the position of the (1)L(b) origin and of the fluorescence maximum, which are largely red-shifted with respect to the gas phase or to solutions in apolar solvents. A large, and increasing with pressure, frequency difference between the (1)L(b) origin and the blue edge of the fluorescence spectrum suggests that the emitting state is (1)L(a), that is known to be more stabilized than (1)L(b) by dipolar relaxation. Crystalline indole was found to be very stable with respect to pressure-induced reactivity. Only traces of a reaction product, containing saturated C-H bonds, are detected after a full compression-decompression cycle. In addition, differently from many unsaturated compounds at high pressure, irradiation with light matching a two-photon absorption for a HOMO-LUMO transition has no enhancing effect on reactivity. The chemical stability of indole at high pressure is ascribed to the crystal structure, where nearest neighbor molecules, formig H-bonds, are not in a favorable position to react, while reaction between equivalent molecules, for which a superposition of the pi electron clouds would be possible, is hindered by H-bonded molecules. Consistently, no excimer emission was observed except at the cell opening at the end of the compression-decompression run. Extremely limited chemical reactivity and excimer formation likely occur at crystal defects, evidencing the strict connection between the two phenomena.

Two-Photon Excitation Spectroscopy of Carotenoid-Containing and Carotenoid-Depleted LH2 Complexes from Purple Bacteria

We applied two-photon fluorescence excitation spectroscopy to LH2 complex from purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides . Bacteriochlorophyll fluorescence was measured under two-photon excitation of the samples within the 1200-1500 nm region. Spectra were obtained for both carotenoid-containing and -depleted complexes of each bacterium to allow their direct comparison. The depletion of carotenoids did not alter the two-photon excitation spectra of either bacteria. The spectra featured a wide excitation band around 1350 nm (2x675 nm, 14,800 cm(-1)) which strongly resembled two-photon fluorescence excitation spectra of similar complexes published by other authors. We consider obtained experimental data to be evidence of direct two-photon excitation of bacteriochlorophyll excitonic states in this spectral region.

Synthesis of two carbazole-based dyes and application of two-photon initiating polymerization

Two carbazole-based polymerization initiators possessing blue fluorescence emission have been synthesized via Wittig reaction in the solid phase at room temperature. Two-photon excited fluorescence (TPEF) spectra for them were investigated under 800 nm fs laser pulse and two-photon absorption cross sections were determined by the Z-scan technique. Then two-photon initiating polymerization (TPIP) microfabrication experiments were successfully carried out. Three-dimensional lattice and artificial defects were gained, indicating that they were viable candidates for the two-photon polymerization initiator in practical application of microfabrication.

Spectroscopic study on the structural isomers of 7-azaindole(ethanol)n (n=1–3) and multiple-proton transfer reactions in the gas phase

The resonance-enhanced two-photon ionization (RE2PI) and laser-induced fluorescence excitation spectra were recorded for the S(1)-S(0)(pipi( *)) region of the 7-azaindole(ethanol)(n) (n=1-3) [7AI(EtOH)(n) (n=1-3)] clusters in the gas phase to investigate the geometrical structures and the multiple-proton/hydrogen atom transfer reaction dynamics. Four and two structural isomers were identified for 7AI(EtOH)(2) and 7AI(EtOH)(3), respectively. Density functional theory calculations at the B3LYP/6-31++G( * *)/6-31G( *) level predicted four different conformations of the ethyl group for 7AI(EtOH)(2), in good agreement with the observation of the four structural isomers in the RE2PI spectra. Visible fluorescence from the tautomeric forms was observed in the S(1) states for all isomers of 7AI(EtOH)(2), but no sign of double-proton/hydrogen atom transfer and quadruple-proton/hydrogen atom transfer has been obtained in the electronic spectra of 7AI(EtOH)(1) and 7AI(EtOH)(3), respectively. These results suggest that the multiple-proton transfer reaction is cluster-size selective, and the triple-proton/hydrogen atom transfer potential is dominated by the cyclic hydrogen-bonded network in 7AI(EtOH)(2). The excitation of the in-phase intermolecular stretching vibration prominently enhances the excited-state triple-proton/hydrogen atom transfer reaction.

The facile synthesis of novel phenothiazine derivatives for blue, yellow-green, and red light emission

Two blue emitters, 3,7-bis(5′-(trifluoromethyl)-1′H-pyrazole-3′-yl)10-ethylphenothiazine and 3,7-bis(5′-(phenyl)-1′H-pyrazole-3′-yl)10-ethyl-phenothiazine and one red, bis-dioxaborine emitter, each based on two, yellow-green emitting, bis-β-diketonate, phenothiazine derivatives, were obtained in high yield and characterized. Systematic investigation of the two-photon absorption characteristics was carried out using one-photon absorption, one-photon excited fluorescence as well as two-photon excited fluorescence spectra. TDDFT computational studies were undertaken to secure an insight into the nature of the compounds' electrochemical and photophysical properties. Low-energy absorption transmissions resemble those observed for the experimental one-photon absorption spectra.

Delivery of sub-10-fs pulses for nonlinear optical microscopy by polarization-maintaining single mode optical fiber

Broadband, sub-10-fs pulses, can be propagated through polarization-maintaining single mode fiber (PMF) for use in nonlinear optical microscopy (NLOM). We demonstrate delivery of near transform-limited, 1 nJ pulses from a Ti:Al(2)O(3) (75 MHz repetition rate) oscillator via PMF to the NLOM focal plane while maintaining 120 nm of bandwidth. Negative group delay dispersion (GDD) introduced to pre-compensate normal dispersion of the optical fiber and microscope optics ensured linear pulse propagation through the PMF. The minimized time-bandwidth product of the laser pulses at the NLOM focus allowed the nonlinear excitation of multiple fluorophores simultaneously without central wavelength tuning. Polarization sensitive NLOM imaging using second harmonic generation in collagen was demonstrated using PMF delivered pulses. Two-photon excited fluorescence spectra and second harmonic images taken with and without the fiber indicates that the fiber based system is capable of generating optical signals that are within a factor of two to three of our traditional NLOM.

Two-Photon Absorption and Excited State Dynamics of Two Organic Molecules Containing Donor/Acceptor Moieties

Two new two-photon absorption (TPA) molecules, named SK-G1 and NT-G1, are synthesized and the photo-physical characteristics are investigated by using linear absorption spectra, one-photon fluorescence spectra and two-photon excited fluorescence spectra. Both the compounds exhibit TPA properties, and the TPA values determined by z-scan measurement are 10GMand 39GMfor SK-G1 and NT-G1, respectively, at wavelength 800nm. Time-resolved spectroscopic techniques are employed to further explore the excited state dynamics of NT-G1with larger TPA cross section. The research results show that there is an ultrafast intraband energy transfer process (about 3ps) before the formation of charge transfer state with a relatively long lifetime.

Optical properties of stilbene-type dyes containing various terminal donor and acceptor groups

A series of “D-π-A” stilbene-type dyes, named as trans-4-[p-(N,N-hydroxyethyl) aminino-styryl]-N-methylpyridinium iodide (DHEASPI-C1), trans-4-[p-(N,N-hydroxyethyl) aminino-styryl]-N-butylpyridinium bromide (DHEASPBr-C4), trans-4-[p-(N,N-hydroxyethyl) aminino-styryl]-N-octylpyridinium bromide (DHEASPBr-C8) and trans-4-[p-(N,N-hydroxyethyl) aminino-styryl]-N-dodecylpyridinium bromide (DHEASPBr-C12), respectively, have been synthesized and their optical properties have been experimentally investigated. When DHEASPI-C1 are compared with trans-4-[p-(N,N-diethylamino) styryl]-N-methylpyridinium iodide (DEASPI) and trans-4-[p-(N-hydroxyethyl-N-ethylamino) styryl]-N-methylpyridinium iodide (HEASPI), both the electronegativity of two hydroxyl and intra-molecular hydrogen bond decrease the donor ability of the ethyl chain, there are obvious blue shifts both in single-photon absorption spectra, fluorescence spectra and two-photon excited fluorescence spectra. Interestingly, fluorescence intensity of DHEASPI-C1 is the biggest. There are little shifts from DHEASPBr-C4 to DHEASPBr-C8 and to DHEASPBr-C12 in their spectra. As dyes’ two-photon excited fluorescence spectra were concerned, pumped by 1064nm, <130fs mode-locked Nd:YAG laser, their peak locations were between 613 and 623nm.