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We report on the optoelectronic properties of a series of unsymmetrical π-conjugated phenyleneethynylene macromolecules bearing ferrocene (Fc) as the electron-donor group (D), (benzyl) benzoate (Bz) or benzoic acid (Ac) as the electron attractor group (A) and connected through 2,5-di(alcoxy) phenyleneethynylene(s) (

A two-step route to strongly absorbing and efficiently orange to deep red fluorescent, doubly B/N-doped, ladder-type pyrrolo[3,2-b]pyrroles has been developed. We synthesize and study a series of derivatives of these four-coordinate boron-containing, nominally quadrupolar materials, which mostly exhibit one-photon absorption in the 500–600 nm range with the peak molar extinction coefficients reaching 150 000, and emission in the 520–670 nm range with the fluorescence quantum yields reaching 0.90. Within the family of these ultrastable dyes even small structural changes lead to significant variations of the photophysical properties, in some cases attributed to reversal of energy ordering of alternate-parity excited electronic states. Effective preservation of ground-state inversion symmetry was evidenced by very weak two-photon absorption (2PA) at excitation wavelengths corresponding to the lowest-energy, strongly one-photon allowed purely electronic transition. π-Expanded derivatives and those possessing electron-donating groups showed the most red-shifted absorption- and emission spectra, while displaying remarkably high peak 2PA cross-section (σ2PA) values reaching ∼2400 GM at around 760 nm, corresponding to a two-photon allowed higher-energy excited state. At the same time, derivatives lacking π-expansion were found to have a relatively weak 2PA peak centered at ca. 800–900 nm with the maximum σ2PA ∼50–250 GM. Our findings are augmented by theoretical calculations performed using TD-DFT method, which reproduce the main experimental trends, including the 2PA, in a nearly quantitative manner. Electrochemical studies revealed that the HOMO of the new dyes is located at ca. −5.35 eV making them relatively electron rich in spite of the presence of two B−–N+ dative bonds. These dyes undergo a fully reversible first oxidation, located on the diphenylpyrrolo[3,2-b]pyrrole core, directly to the di(radical cation) stage., Ladder-type heterocycles encompassing two B−–N+ dative bonds possess intense green to red emission, large 2PA cross-sections and superb photostability.

The Laporte rule dictates that one- and two-photon absorption spectra of inversion-symmetric molecules should display alternatively forbidden electronic transitions; however, for organic fluorophores, drawing clear distinction between the symmetric- and non-inversion symmetric two-photon spectra is often obscured due to prevalent vibronic interactions. We take advantage of consecutive single- and double-protonation to break and then reconstitute inversion symmetry in a nominally symmetric diketopyrrolopyrrole, causing large changes in two-photon absorption. By performing detailed one- and two-photon titration experiments, with supporting quantum-chemical model calculations, we explain how certain low-frequency vibrational modes may lead to apparent deviations from the strict Laporte rule. As a result, the system may be indeed considered as an on-off-on inversion symmetry switch, opening new avenues for two-photon sensing applications.

Lipophilic fluorophores are widely implemented in nonlinear microscopy; however, few existing membrane-specific probes combine the high brightness of two-photon excited fluorescence (2PEF) with pH sensitivity. Herein we describe four novel two-photon excited fluorophores, based on a coumarin 151 core structure, where lipophilicity is induced by a covalently attached phosphazene moiety. Changing the environmental acidity using trifluoromethanesulfonic (triflic) acid leads to profound changes in the linear fluorescence and 2PEF characteristics, due to chromophores' switching between neutral- and protonated forms. We characterize this dependence by measuring the two-photon absorption (2PA) spectra over the region λ

A series of cis-platinum(II) acetylide complexes containing two-photon-absorbing chromophores have been synthesized and characterized to explore the effects of stereochemistry on the nonlinear absorption properties. The molecules feature 4-(phenylethynyl)phenylethynylene (PE2), diphenylaminofluorene (DPAF), and benzothiazolylfluorene (BTF) ligands. The photophysical properties were investigated under one- and two-photon conditions and compared to the known trans analogues via UV-visible absorption, photoluminescence, femtosecond and nanosecond transient absorption (TA), nanosecond z-scan, and femtosecond two-photon absorption (2PA). The bent cis complexes exhibit blue shifts in the absorption, emission, femtosecond, and nanosecond TA spectra along with lower molar extinction coefficients and lower phosphorescence yields relative to the trans complexes suggesting less efficient Pt-induced spin-orbit coupling and intersystem crossing in the cis configuration. The cis chromophores are noncentrosymmetric and therefore show dipolar behavior with a pronounced 2PA in the 0-0 transition of the S0 → S1 band, while the trans complexes show quadrupolar behavior with a forbidden 0-0 transition. In the S0 → Sn region, both cis and trans complexes show intense two-photon-absorption bands (up to 3700 GM by the peak cross section for cis-BTF) which contain a significant contribution from the excited state absorption (S1 → Sn). All six complexes exhibit comparable nonlinear absorption response with a significant contribution from triplet-triplet absorption that slightly favors trans complexes but is more strongly dependent upon the structure of the π-conjugated chromophore.

Two-photon excitation of fluorescence (2PEF) is a versatile tool for high-resolution functional microscopy, but few existing fluorophores combine high two-photon brightness with ability to report on key in vivo- and in vitro environmental factors such as pH, ionic strength, or solvent polarity. We describe four novel pH-sensitive derivatives of coumarin 151 with different attached phosphazene moieties, offering high lipophilicity both in their neutral- and protonated forms at near physiological conditions (neutral to mildly basic). We measure the two-photon absorption (2PA) and 2PEF emission spectra, as well as the fluorescence quantum yields in neutral acetonitrile solution, as well as with the addition of small amount of trifluoromethanesulfonic (triflic) or hydrochloric acid. We show that the neutral form has high two-photon brightness in the basic environments, whereas protonation quenches the fluorescence by a factor of ~ 20, while blueshifting the absorption- and emission peaks by 60 nm. By evaluating the ratio of the measured 2PA and 1PA spectra corresponding to the transition to the lowest excited singlet state S1 we show, for the first time, that the permanent molecular dipole moment change is reduced upon the protonation. This conclusion is supported by our quantum-chemical calculations which indicate that protonation leads to reduction of extent of conjugation in the system. We also measure the time-dependent fluorescence and show that in neutral environment the fluorescence displays a single-exponential decay, while upon protonation the decay is distinctly non-single-exponential in character.

Change of the permanent molecular electric dipole moment, Δμ, in a series of nominally centrosymmetric and noncentrosymmteric ferrocene-phenyleneethynylene oligomers was estimated by measuring the two-photon absorption cross-section spectra of the lower energy metal-to-ligand charge-transfer transitions using femtosecond nonlinear transmission method and was found to vary in the range up to 12 D, with the highest value corresponding to the most nonsymmetric system. Calculations of the Δμ performed by the TD-DFT method show quantitative agreement with the experimental values and reveal that facile rotation of the ferrocene moieties relative to the organic ligand breaks the ground-state inversion symmetry in the nominally symmetric structures.

We use TD-DFT to calculate the one-photon absorption (1PA) and two-photon absorption (2PA) properties of C153 and Prodan in toluene and DMSO, and benchmark different methods relative to accurate experimental data available from the literature on these particular systems. As the first step, we modify the range-separated TD-DFT to provide the best prediction for the peak 1PA wavelength, and then apply the optimized functionals to achieve quantitative predictions of the corresponding two-photon absorption cross section, σ2PA, with an accuracy ∼10-20% in C153 and ∼20-30% in Prodan. To elucidate the origin of residual discrepancies between the theory and experimental observations, we invoked the two essential states model for σ2PA, which allows us to verify not only the transition wavelength and the σ2PA value, but also to quantitatively benchmark the calculation of key molecular parameters such as the transition dipole moment and the change of the permanent dipole moment. Such comprehensive cross-checking indicates that a larger discrepancy in Prodan is most likely caused by a noted failure of DFT to predict the relative intensity and relative ordering of closely lying excited states with different degrees of intramolecular charge transfer, which we further support by analyzing the predictions obtained by high-level coupled-cluster calculations in the gas phase. Our results highlight the utility of benchmarking the calculations not only relative to other theoretical methods, but also in comparison to the experimental measurements, wherever such data are available.

Origin of the initial charge separation in optically-excited Ruthenium(II) tris(bidentate) complexes of intrinsic D3 symmetry has remained a disputed issue for decades. Here we measure the femtosecond two-photon absorption (2PA) cross section spectra of [Ru(2,2′-bipyridine)3]2 and [Ru(1,10-phenanthroline)3]2 in a series of solvents with varying polarity and show that for vertical transitions to the lower-energy 1MLCT excited state, the permanent electric dipole moment change is nearly solvent-independent, Δμ = 5.1–6.3 D and 5.3–5.9 D, respectively. Comparison of experimental results with quantum-chemical calculations of complexes in the gas phase, in a polarizable dielectric continuum and in solute-solvent clusters containing up to 18 explicit solvent molecules indicate that the non-vanishing permanent dipole moment change in the nominally double-degenerate E-symmetry state is caused by the solute-solvent interaction twisting the two constituent dipoles out of their original opposite orientation, with average angles matching the experimental two-photon polarization ratio. Ruthenium(II) complexes are widely used as photosensitisers for electron transfer, but given their high intrinsic symmetry, the origin of the excited state dipole moment is unclear. Here two-photon absorption experiments supported by theoretical calculations suggest this arises from solvent-solute electronic interactions.

The spin-lattice relaxation time in the 4 A 2 ground state of C r 3 + in ruby can be on the order of magnitude of seconds at liquid helium temperatures in low magnetic fields B | | c . This allows for highly efficient optical pumping of the spin levels in this system. Such pumping can then in turn be employed to drastically increase the optical density of the inhomogeneously broadened R 1 ( ± 3 / 2 ) or R 1 ( ± 1 / 2 ) lines and also to narrow the spectral hole width because Cr–Cr electron-spin flips (cross relaxation) are suppressed in the ground state, leading to longer dephasing times T 2 ∗ . Both effects can be employed to further reduce the group velocity of light pulses propagating through a transient spectral hole.

Formation of optical solitons in self-induced transparency (SIT) regime, where light pulses propagate virtually without loss in nominally strongly absorbing medium, is one of the most striking coherent transient phenomena in optics. Here we study experimentally and by numeric simulations how a square shape pulse gradually transforms into a smooth sech shape pulse of well-defined pulse area, depending on the parameters such as the pulse amplitude, duration, propagation distance etc. The SIT experiments for circularly polarized light are performed in the R1−3∕2) line of a 30 ppm ruby (α-Al2O3:Cr3+) at 1.7 K in a magnetic field of BIIc = 4.5 T, which corresponds to effective absorption coefficient, αL =14.5. In such a magnetic field and temperature range, a 30 ppm ruby is in the so-called superhyperfine limit resulting in a very long decoherence (phase memory) time, TM = 50 μs. We show, in good quantitative agreement with the simulations, how SIT soliton is formatted and how this results in extremely slow pulse peak propagation velocity of ∼300 m∕s, which is to date, the slowest pulse propagation ever observed in a SIT experiment. We also show that for accurate quantitative description of the observed SIT and soliton pulse shapes, the simulations need to account for variation of the incident pulse amplitude across the beam spatial profile. Potential implications of the SIT effect on classical- and quantum information storage will be discussed.

Fluorescence excited by instantaneous three-photon absorption (3PA) in organic fluorophores is gaining importance as a versatile modality for deep-tissue microscopy and imaging. However, due to technical difficulty of quantifying the higher-order nonlinear absorption cross-section, reliable 3PA cross section values, σ3PA, covering a broad spectral range have been so far not available. This lack of experimental data hinders us from gaining quantitative understanding of relevant structure-property relationships as well as impedes progress towards developing 3-photon fluorophores optimized for various applications. We report on measurement of the absolute 3PA cross section spectra in the 950 – 1750 nm range in a series of common organic fluorophores in various solvents: (a) Rhodamine 6G in deuterated methanol, (b) Coumarin 153 in DMSO and toluene, (c) Prodan in DMSO and toluene, (d) Fluorescein in pH11 buffer, (e) AF455 in toluene, (f) BDPAS in deuterated methylene chloride. In these experiments, we employ femtosecond wavelength-tunable optical parametric amplifier to excite fluorescence signal that has cubic dependence on the incident photon flux. Absolute values of σ3PA are determined using two complementary methods: (i) calibrating the fluorescence signal relative to one-photon (linear) excitation combined with accurate measurement of the pulse temporal- and spatial profile to determine the excitation photon flux and (ii) calibration of the cubic fluorescence signal relative to quadratic florescence excited in fluorophores with known two-photon absorption cross section. Depending on the method utilized, the peak σ3PA values have estimated accuracy 50% and vary in the range, σ3PA = 10-81 - 10-79 cm6 s2 photon-2 , depending on the system studied, with AF455 showing the most enhanced 3PA efficiency. The 3PA spectral shapes have estimated accuracy of 20% and show some unexpected deviations from corresponding one-photon spectral profiles.

Degenerate instantaneous two-photon absorption (2PA) cross section and 2PA spectrum measurements were used to determine the molecular electric dipole change in the metal-to-ligand charge-transfer transition of ruthenium(II) triscomplexes of 2,2’-bipyridine and 1,10-phenanthroline ligands. Comparison between the 2PA and one-photon absorption (1PA) spectra indicate that the phenanthroline complex has D3 symmetry in the ground state, while the bipyridine complex has its symmetry lowered. The high accuracy of the nonlinear cross section measurements was achieved by means of improved 2PA reference standards, and the dipole change was evaluated using the two essential states model of 2PA applied in the lowest-energy transitions. We demonstrate that quantitative 2PA spectroscopy is a viable alternative to standard methods used to estimate change of molecular dipole moment, and is uniquely informative of the effects of different solvents and local environments on the absorber.

Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP—the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.

A series of platinum(II) acetylide complexes containing p-phenylenevinylene and moieties end-capped with triphenylamine groups have been incorporated into poly(methyl methacrylate) (PMMA) monoliths for optical power limiting applications. The one- and two-photon photophysical properties were investigated and compared to the photophysical properties in THF. The absolute two-photon absorption cross-section values for the monolith samples were measured and are comparable to the values obtained in solution. In the PMMA monoliths, the complexes retained the important two-photon absorption and reverse saturable absorption properties necessary for optical power limiting via dual mode mechanism, and their strong nonlinear absorption property was demonstrated by the open-aperture Z-scan method. Photostability studies of the p-phenylenevinylene platinum(II) acetylide complexes showed two photodegradation processes: a trans-to-cis isomerization and a singlet-oxygen sensitized self-oxidative cleavage. The photostability of the least photostable complex TPV0 was increased upon incorporation into a PMMA matrix.

We present a new approach for determining the strength of the dipolar solute-induced reaction field, along with the ground- and excited-state electrostatic dipole moments and polarizability of a solvated chromophore, using exclusively one-photon and two-photon absorption measurements. We verify the approach on two benchmark chromophores N,N-dimethyl-6-propionyl-2-naphthylamine (prodan) and coumarin 153 (C153) in a series of toluene/dimethyl sulfoxide (DMSO) mixtures and find that the experimental values show good quantitative agreement with literature and our quantum-chemical calculations. Our results indicate that the reaction field varies in a surprisingly broad range, 0-10(7) V cm(-1) , and that at close proximity, on the order of the chromophore radius, the effective dielectric constant of the solute-solvent system displays a unique functional dependence on the bulk dielectric constant, offering new insight into the close-range molecular interaction.

An improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, is introduced, called hmKeima8.5. Its increased intracellular brightness and long Stokes shift (~180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Here we demonstrate its utility in intracellular fluorescence correlation spectroscopy applications., We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein–protein interactions. We also use MPE-FCCS to detect drug–protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.

Fluorescent DNA base analogs and intrinsic fluorophores are gaining importance for multiphoton microscopy and imaging, however, their quantitative nonlinear excitation properties have been poorly documented. Here we present the two-photon absorption (2PA) spectra of 2-aminopurine (2AP), 7-methyl guanosine (7MG), isoxanthopterin (IXP), 6-methyl isoxanthopterin (6MI), as well as L-tryptophan (L-trp) and 3-methylindole (3MI) in aqueous solution and some organic solvents measured in the wavelength range 550 - 810 nm using femtosecond two-photon excited fluorescence (2PEF) and nonlinear transmission (NLT) methods. The peak 2PA cross section values range from 0.1 GM (1 GM = 10−50 cm4 s photon−1) for 2AP to 2.0 GM for IXP and 7MG. Assuming typical excitation conditions for a scanning 2PEF microscope, we estimate a maximum image frame rate of ~175 frames per second (FPS).

International audience; Five centrosymmetric and one dipolar pyrrolo[3,2-b]pyrroles, possessing either two or one strongly electron-withdrawing nitro group have been synthesized in a straightforward manner from simple building blocks. For the symmetric compounds, the nitroaryl groups induced spontaneous breaking of inversion symmetry in the excited state, thereby leading to large solvatofluorochromism. To study the origin of this effect, the series employed peripheral structural motifs that control the degree of conjugation via altering of dihedral angle between the 4-nitrophenyl moiety and the electron-rich core. We observed that for compounds with a larger dihedral angle, the fluorescence quantum yield decreased quickly when exposed to even moderately polar solvents. Reducing the dihedral angle (i.e., placing the nitrobenzene moiety in the same plane as the rest of the molecule) moderated the dependence on solvent polarity so that the dye exhibited significant emission, even in THF. To investigate at what stage the symmetry breaking occurs, we measured two-photon absorption (2PA) spectra and 2PA cross-sections (σ2PA ) for all six compounds. The 2PA transition profile of the dipolar pyrrolo[3,2-b]pyrrole, followed the corresponding one-photon absorption (1PA) spectrum, which provided an estimate of the change of the permanent electric dipole upon transition, ≈18 D. The nominally symmetric compounds displayed an allowed 2PA transition in the wavelength range of 700-900 nm. The expansion via a triple bond resulted in the largest peak value, σ2PA =770 GM, whereas altering the dihedral angle had no effect other than reducing the peak value two- or even three-fold. In the S0 →S1 transition region, the symmetric structures also showed a partial overlap between 2PA and 1PA transitions in the long-wavelength wing of the band, from which a tentative, relatively small dipole moment change, 2-7 D, was deduced, thus suggesting that some small symmetry breaking may be possible in the ground state, even before major symmetry breaking occurs in the excited state

With the goal of elucidating electronic and conformational effects on structure–spectroscopic property relationships in platinum acetylides, we synthesized a series of nominally centrosymmetric chromophores trans-Pt(PBu3)2(C≡C-Phenyl-X)2, where X = diphenylamino (DPA), NH2, OCH3, t-Bu, CH3, H, F, benzothiazole (BTH), CF3, CN, and NO2. We collected one- and two-photon absorption spectra and also performed density functional theory (DFT) and time-dependent (TD) DFT calculations on the ground- and excited-state properties of these compounds. The DFT calculations revealed facile rotation between the two ligands, suggesting that the compounds exhibit nonplanar ground-state conformations in solution. TDDFT calculation of the S1 state energy and transition dipole moment for a nonplanar conformation gave good agreement with experiment. Two-photon absorption spectra obtained from these compounds allowed estimation of the change of permanent electric dipole moment upon vertical excitation from ground state to S1 st...

Two-photon absorption (2PA) cross sections for 2-aminopurine (2AP), 7-methyl guanosine (7MG), 6-methyl isoxanthopterin (6MI), isoxanthopterin (IXP), 3-methylindole (3MI) and tryptophan (L-trp) are measured for excitation wavelengths between 550 and 810 nm using two independent methods: femtosecond two-photon excited fluorescence (2PEF) and femtosecond nonlinear transmission (NLT). For all systems we find that in the long wavelength region of the lowest-energy one-photon allowed singlet-singlet transition the 2PA spectral profiles follow the one-photon absorption spectral profiles, thus indicating the strongly dipolar nature of the transition. We use the measured 2PA cross sections to estimate the corresponding permanent electric dipole moment change and find the values to vary in the range 1 to 5 D. These experimental findings are corroborated by quantum-chemical computations.

Fluorescence induced by 1wo-photon absorption (2PA) and three-photon absorption (3PA) is becoming an increasingly important tool for deep-tissue microscopy, especially in conjunction with genetically-encoded functional probes such as fluorescent proteins (FPs). Unfortunately, the efficacy of the multi-photon excitation of FPs is notoriously low, and because relations between a biological fluorophore’s nonlinear-optical properties and its molecular structure are inherently complex, there are no practical avenues available that would allow boosting the performance of current FPs. Here we describe a novel method, where we apply directed evolution to optimize the 2PA properties of EGFP. Key to the success of this approach consists in high-throughput screening of mutants that would allow selection of variants with promising 2PA and 3PA properties in a broad near-IR excitation range of wavelength. For this purpose, we construct and test a wide field-of-view (FOV), femtosecond imaging system that we then use to quantify the multi-photon excited fluorescence in the 550- 1600 nm range of tens of thousands of E. coli colonies expressing randomly mutated FPs in a standard 10 cm diameter Petri dish configuration. We present a quantitative analysis of different factors that are currently limiting the maximum throughput of the femtosecond multi-photon screening techniques and also report on quantitative measurement of absolute 2PA and 3PA cross sections spectra.

Change of permanent electric dipole moment in the lower-energy charge transfer transitions for a series of symmetrical and non-symmetrical ferrocene-phenyleneethynylene oligomers were studied by measuring the corresponding femtosecond two-photon absorption cross section spectra, and were determined to be in the range Δμ = 3 – 10 D. Quantum-chemical calculations of Δμ for the non-symmetrical oligomers show good quantitative agreement with the experimental results, thus validating two-photon absorption spectroscopy as a viable experimental approach to study electrostatic properties of organometallics and other charge transfer systems.

Trans- A 2 B 2-tetrakis(arylethynyl)porphyrins with suitable solubility in CH 2 Cl 2, CHCl 3, EtOAc , acetone and toluene have been obtained for the first time. Among two possible strategies the one comprising the synthesis of 5,15-dibromo-10,20-bis[(isopropylsilyl)ethynyl]porphyrin proved to be more efficient. The pathway towards densely substituted arylacetylenes has been optimized. The use of previously identified 3,4,5-trialkoxyaryl substituent was crucial for achieving the reasonable solubility. The optical properties of meso-substituted tetrakis(arylethynyl)porphyrins were studied showing that strong polarization imparted by direct conjugation of all four substituents with porphyrin core resulted not only in strong absorption of red light but also in a relatively long triplet lifetime. Meso-tetrakis(arylethynyl)porphyrins have a significantly longer lifetime of T1 state than bis(arylethynyl)porphyrins and in their case all the states are mixtures of transitions between the HOMO-1, HOMO and LUMO, LUMO+1 MOs. We show that the presence of two additional arylethynyl substituents at meso-positions enhance the maximum two-photon absorption cross-section of trans- A 2 B 2-tetrakis(arylethynyl)porphyrins by more than one order of magnitude. Maximum values as high as σ2 = 500 GM at 950 nm result from realization of suitable conditions for effective resonance enhancement along with a lowering of the energy and intensification of the two-photon allowed transitions in the Soret region.

In order to understand the photophysics and non-linear optical properties of carbazole containing π-conjugated oligomers of the type ET-Cbz-TE (E = ethynylene, T = 2,5-thienylene, Cbz = 3,6-carbazole), a detailed investigation was carried out on a series of oligomers that feature Au(i) or Pt(ii) acetylide "end groups", as well as a Pt(ii)-acetylide linked polymer (CBZ-Au-1 and CBZ-Pt-1, CBZ-Poly-Pt). These organometallic chromophores were characterized by UV-visible absorption and variable temperature photoluminescence spectroscopy, nanosecond transient absorption spectroscopy, open aperture nanosecond z-scan and two photon absorption (2PA) spectroscopy. The Au(i) and Pt(ii) oligomers and polymer exhibit weak fluorescence in fluid solution at room temperature. Efficient phosphorescence is observed from the Pt(ii) systems below 150 K in a solvent glass; however, the Au(i) oligomer exhibits only weak phosphorescence at 77 K. Taken together, the emission results indicate that the intersystem crossing efficiency for the Pt(ii) chromophores is greater than for the Au(i) oligomer. Nonetheless, nanosecond transient absorption indicates that direct excitation affords moderately long-lived triplet states for all of the chromophores. Open aperture z-scan measurement shows effective optical attenuation can be achieved by using these materials. The 2PA cross section in the degenerate S0→S1 transition region was in the range 10-100 GM, and increased monotonically toward shorter wavelengths, reaching 800-1000 GM at 550 nm.

Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

We study instantaneous two-photon absorption (2PA) in a series of nominally quasi-centrosymmetric trans-bis(tributylphosphine)-bis-(4-((9,9-diethyl-7-ethynyl-9H-fluoren-2-yl) ethynyl)-R)-platinum complexes, where 11 different substituents, R = N(phenyl)2(NPh2), NH2, OCH3, t-butyl, CH3, H, F, CF3, CN, benzothiazole, and NO2, represent a range of electron-donating (ED) and electron-withdrawing (EW) strengths, while the Pt core acts as a weak ED group. We measure the 2PA cross section in the 540–810 nm excitation wavelength range by complementary femtosecond two-photon excited fluorescence (2PEF) and nonlinear transmission (NLT) methods and compare the obtained values to those of the Pt-core chromophore and the corresponding noncentrosymmetric side group (ligand) chromophores. Peak 2PA cross sections of neutral and ED-substituted Pt complexes occur at S0 → Sn transitions to higher energy states, above the lowest-energy S0 → S1 transition, and the corresponding values increase systematically with increasing E...

Background Broader clinical acceptance of photodynamic therapy is currently hindered by (a) poor depth efficacy, and (b) predisposition towards establishment of an angiogenic environment during the treatment. Improved depth efficacy is being sought by exploiting the NIR tissue transparency window and by photo-activation using two-photon absorption (2PA). Here, we use two-photon activation of PDT sensitizers, untargeted and targeted to SST2 receptors or EGF receptors, to achieve deep tissue treatment. Methods Human tumor lines, positive or negative for SST2r expression were used, as well as murine 3LL cells and bovine aortic endothelial cells. Expression of SST2 receptors on cancer cells and tumor vasculature was evaluated in vitro and frozen xenograft sections. PDT effects on tumor blood flow were followed using in vivo scanning after intravenous injection of FITC conjugated dextran 150 K. Dependence of the PDT efficacy on the laser pulse duration was evaluated. Effectiveness of targeting to vascular SST2 receptors was compared to that of EGF receptors, or no targeting. Results Tumor vasculature stained for SST2 receptors even in tumors from SST2 receptor negative cell lines, and SST2r targeted PDT led to tumor vascular shutdown. Stretching the pulse from ~ 120 fs to ~ 3 ps led to loss of the PDT efficacy especially at greater depth. PDT targeted to SST2 receptors was much more effective than untargeted PDT or PDT targeted to EGF receptors. General significance The use of octreotate to target SST2 receptors expressed on tumor vessels is an excellent approach to PDT with few recurrences and some long term cures.

The synthesis of a 2,5-bis(dodecanoxy)phenyleneethynylene-butadiyne series with 2, 4, 6 and 8 phenyl rings is reported. Sonogashira coupling reaction, rather than the Glaser/Eglinton/Hay reactions classically used for butadiyne formation was applied. The molecular structures of all compounds were confirmed by 1H, DEPT-135, APT 13C, MALDI-TOF, FTIR and FT-Raman analyses. The linear and nonlinear optical properties were studied in solution by UV/Vis, static and time-resolved fluorescence, and by two-photon absorption (2PA) spectroscopy. With the exception of the dimer, for which intersystem crossing is very favoured due to the low energy gap between the singlet and triplet states as theoretically predicted, the other oligomers present high fluorescence quantum yields (0.77–0.82) and large cross-sections (up to 5000 GM for the octamer) that could be applied in multiphoton microscopy or nonlinear optics.

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca2+) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca2+ indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca2+ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

The linear and nonlinear optical properties of a series of linear and cross-conjugated platinum(II) acetylide complexes that contain extended p-(phenylene vinylene) chromophores are reported. The complexes exhibit very high femtosecond two-photon absorption (2PA) cross section values (σ(2) up to 10,000 GM), as measured by nonlinear transmission (NLT) and two-photon excited fluorescence (2PEF) methods. The large 2PA cross sections span a broad range of wavelengths, 570-810 nm, and they overlap with the triplet excited state absorption. Spectral coincidence of high cross section 2PA and triplet absorption is a key feature giving rise to efficient dual-mode optical power limiting (OPL).

A pseudo-symmetric two-photon absorbing dye (1) containing a central piperazine unit substituted with (benzothiazol-2- yl)-9,9-diethylfluoren-2-yl pendant groups has been synthesized and characterized. The molecule has a two-photonabsorption cross-section of σ2 = 140 GM in tetrahydrofuran at ~ 740 nm and shows significant solvatochromism in the excited-state fluorescence spectra. The emission spectra broaden and the maxima bathochromically shift from 411 nm to 524 nm in n-hexane and acetonitrile, respectively. Moreover, the central piperazine moiety serves as a potential chelation site for ions. Addition of copper(I) hexafluorophosphate and zinc(II) triflate in acetonitrile indicate ground-state complexation with a shift in the emission maximum from 524 nm to 489 nm and 487 nm, respectively. Interestingly, the newly formed Cu and Zn complexes are more strongly emissive than the free dye. Finally, addition of p-toluenesulfonic acid in tetrahydrofuran also blue-shifts the emission maximum, but the intensity is quenched. Due to the photophysical changes induced by addition of metal ions and protons, the dye shows promise as a potential sensor.

We report femtosecond light-induced macromolecular self-assembly (FLIMSA), which is observed when a high peak intensity femtosecond laser beam propagates through aqueous solution of pseudoisocyanine iodide (PIC) J-aggregates and induces the formation of 0.1 – 1.0 mm-size tube-like structure surrounding the laser beam, while at the same time allowing the beam to continue propagating without obstruction or scattering. The FLIMSA material is morphologically heterogeneous and gel-like and is formed at the margins rather than at the center of the beam. As a potential explanation of this effect we assume that the FLIMSA is induced by the high photon flux gradient characteristic of the femtosecond laser beam periphery. This hypothesis is corroborated by control experiments, where J-aggregate samples were illuminated with nanosecond laser sources with a varying pulse duration, power- and beam shape characteristics, but where no FLIMSA formation was observed.

Degenerate two-photon absorption (2PA) of a series of organic fluorophores is measured using femtosecond fluorescence excitation method in the wavelength range, λsub2PA/sub= 680-1050 nm, and ~100 MHz pulse repetition rate. The function of relative 2PA spectral shape is obtained with estimated accuracy 5%, and the absolute 2PA cross section is measured at selected wavelengths with the accuracy 8%. Significant improvement of the accuracy is achieved by means of rigorous evaluation of the quadratic dependence of the fluorescence signal on the incident photon flux in the whole wavelength range, by comparing results obtained from two independent experiments, as well as due to meticulous evaluation of critical experimental parameters, including the excitation spatial- and temporal pulse shape, laser power and sample geometry. Application of the reference standards in nonlinear transmittance measurements is discussed.

Optically-acquired fingermarks are widely used as evidence across law enforcement agencies as well as in the courts of law. A common technique for visualizing latent fingermarks on nonporous surfaces consists of cyanoacrylate fuming of the fingerprint material, followed by impregnation with a fluorescent dye, which under ultra violet (UV) illumination makes the fingermarks visible and thus accessible for digital recording. However, there exist critical circumstances, when the image quality is compromised due to high background scattering, high auto-fluorescence of the substrate material, or other detrimental photo-physical and photo-chemical effects such as light-induced damage to the sample. Here we present a novel near-infrared (NIR), two-photon induced fluorescence imaging modality, which significantly enhances the quality of the fingermark images, especially when obtained from highly reflective and/or scattering surfaces, while at the same time reducing photo-damage to sensitive forensic samples.

Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.

The synthesis, one-photon photophysics and two-photon absorption (2PA) of three dipolar D-π-A 4-[9,9-di(2-ethylhexyl)-7-diphenylaminofluoren-2-yl]-2,2':6',2''-terpyridine and their platinum chloride complexes with different linkers between the donor and acceptor are reported. All ligands exhibit (1)π,π* transition in the UV and (1)π,π*/(1)ICT (intramolecular charge transfer) transition in the visible regions, while the complexes display a lower-energy (1)π,π*/(1)CT (charge transfer) transition in the visible region in addition to the high-energy (1)π,π* transitions. All ligands and the complexes are emissive at room temperature and 77 K, with the emitting excited state assigned as the mixed (1)π,π* and (1)CT states at RT. Transient absorption from the ligands and the complexes were observed. 2PA was investigated for all ligands and complexes. The two-photon absorption cross-sections (σ(2)) of the complexes (600-2000 GM) measured by Z-scan experiment are much larger than those of their corresponding ligands measured by the two-photon induced fluorescence method. The ligand and the complex with the ethynylene linker show much stronger 2PA than those with the vinylene linker.

A series of platinum-containing organometallic dimer complexes has been synthesized and the photophysical properties have been investigated under one- and two-photon (2PA) absorption conditions. The complexes have the general structure [DPAF-C[triple bond]C-Pt(PBu(3))(2)-C[triple bond]C-Ar-C[triple bond]C-Pt(PBu(3))(2)-C[triple bond]C-DPAF], where Ar is a pi-conjugated unit, Bu = n-butyl, and DPAF = diphenylamino-2,7-fluorenylene. The core Ar units include 1,4-phenylene, 2,5-thienylene, 5,5'-(2,2'-bithienylene), 2,5-(3,4-ethylenedioxythiophene, 2,1,3-benzothiadiazole, and 4,7-dithien-2-yl-2,1,3-benzothiadiazole. Absorption and photoluminescence spectroscopy indicates that the complexes feature low-lying excited states based on both the core [-Pt(PBu(3))(2)-C[triple bond]C-Ar-C[triple bond]C-Pt(PBu(3))(2)-] chromophore as well as the DPAF units. Photoexcitation of the complexes produces a singlet state excited state, which rapidly undergoes intersystem crossing to afford a triplet state that has a lifetime in the microsecond time domain. In most cases, the lowest energy triplet state is localized on the core chromophore. Femtosecond 2PA spectra are measured along with triplet-triplet absorption spectra and nanosecond intensity-dependent transmission for solutions of the complexes. Each of the complexes features a 2PA absorption band in the near-infrared region (lambda approximately 700-750 nm) with a cross section 50-200 GM that is ascribed to the DPAF chromophore. The complexes also feature broad triplet-triplet absorption throughout the visible and near-infrared regions (lambda approximately 500-800 nm, (TT) approximately 5-10 x 10(4) M(-1) cm(-1)). Each of the complexes exhibits efficient nonlinear absorption of nanosecond pulses in the near-infrared region (600-800 nm), and we demonstrate that effect is most efficient in the chromophores where the 2PA cross section maxima coincides spectrally with the excited triplet state absorption.

We present a new approach to modeling of homogeneous line shape in two-photon absorption (2PA) spectra of chromophores with large permanent dipole moment difference between the ground- and excited electronic states using numerical solution of stochastic two-level density matrix equation of motion. Good agreement with experimental 2PA line shapes is obtained for S 1 ←S 0 transition of Styryl 9 M, which allows us to estimate that the permanent dipole moment difference varies in this chromophore within the S 1 ←S 0 band in the range, Δ μ =12–25 D.

Quantitative knowledge about static molecular electric dipole moments is essential for understanding of intramolecular charge transfer as well as nanometer-scale static electric interactions. However, measuring or determining the molecular electrostatic properties with sufficient accuracy remains a challenging task. In our experiments, we measure the femtosecond two-photon absorption spectra- and cross sections of a range of organic- and organometallic chromophores in solution and use these data to determine the electric dipole moment change in corresponding lowest-energy dipole-allowed transition. Good correspondence of our experimental dipole moments with the quantum-chemical calculations as well as reports by other groups using conventional dipole moment measurement methods suggests that quantitative multiphoton spectroscopy may offer all-optical alternative to the traditional techniques such as Stark effect and electrochromism.

Quantitative two-photon absorption (2PA) cross section and 2PA spectrum measurements were used to determine the molecular electric dipole change in the metal-to-ligand charge-transfer transition of ruthenium(II) tris-complexes of 2,2’-bipyridine ([Ru(bpy)3]2+) and 1,10-phenanthroline ([Ru(phen)3]2+) in several solvents.

We report for the first time two-photon absorption (2PA) cross sections and spectra of corroles within a broad spectral range of excitation wavelengths, 800–1400 nm. Compared to symmetrically-substituted porphyrins, the 2PA spectrum of corroles contains a distinct peak at the laser wavelength close to twice the wavelength of Soret maximum (∼850 nm), with a quite significant cross section, σ2 = 60–130 GM. Lowering of the symmetry relaxes the parity selection rules for 2PA, thus making the Soret band allowed for two-photon transition. We also observe that the strength of the 2PA peak in Soret region strongly decreases with the electron-withdrawing ability of the side substituents.

We study near-IR two-photon absorption (2PA) spectra of nonsymmetric push−pull metal-free phthalocyanines, which are present in solution at room temperature as a mixture of two spectrally nonequivalent tautomers T1 and T2. Large 2PA cross sections, σ2 = (1−3) × 103 GM, are obtained in the excitation wavelength region λex = 815−850 nm and are explained by (1) a new charge transfer state appearing in the one-photon absorption (1PA) spectrum of these compounds at 420−430 nm and (2) a strong resonance enhancement within the quasi-three-level system when the Q-state(s) serve as the real intermediate state(s). In the Q-region, λex = 1100−1500 nm, 2PA and 1PA transitions coincide, and the 2PA strength of any pure electronic or vibronic band is quantitatively described in terms of a two-level approximation.

We demonstrate spatial interference fringe pattern by simultaneous one- and three-photon absorption of UV and near-IR femtosecond pulses in thin film organic solid at room temperature. We use organic dendrimers that are specially designed to have strong fluorescence and very large three-photon absorption cross-section. High fringe visibility allows the quantum interference to be observed by eye.

A combined experimental and theoretical study of the two-photon absorption (2PA) properties of a series of quadrupolar molecules possessing a highly electron-rich heterocyclic core, pyrrolo[3,2-b]pyrrole, is presented. In agreement with quantum-chemical calculations, large 2PA cross-section values, σ2PA ≈10(2) -10(3) GM (1 GM=10(50) cm(4) s photon(-1) ), are observed at wavelengths of 650-700 nm, which correspond to the two-photon allowed but one-photon forbidden transitions. The calculations also predict that increased planarity of this molecule through removal of two N-substituents leads to further increase in the σ2PA values. Surprisingly, the most quadrupolar pyrrolo[3,2-b]pyrrole derivative, containing two 4-nitrophenyl substituents at positions 2 and 5, demonstrates a very strong solvatofluorochromic effect, with a fluorescence quantum yield as high as 0.96 in cyclohexane, whereas the fluorescence vanishes in DMSO.

Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range.

Physics Department, Montana State University, Bozeman, MT 59717, USA Abstract (35 word) Femtosecond 2-photon absorption spectroscopy is used to determine the strength of local electrostatic field in the molecular vicinity of dissolved dipolar chromophores. Experimental techniques facilitating high accuracy 2-photon cross section measurements will be discussed.