Stereochemical Effects on Platinum Acetylide Two-Photon Chromophores.

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 directed evolution of green fluorescent proteins.

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.

Two-photon voltmeter for measuring a molecular electric field.

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.

Polymer Monoliths Containing Two-Photon Absorbing Phenylenevinylene Platinum(II) Acetylide Chromophores for Optical Power Limiting.

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.

Two-photon sensitive protecting groups operating via intramolecular electron transfer: uncaging of GABA and tryptophan.

Improved photo-labile protecting groups, with high sensitivity to two-photon excitation, are needed for the controlled release of drugs, as tools in neuroscience and physiology. Here we present a new modular approach to the design of caging groups based on photoinduced electron transfer from an electron-rich two-photon dye to an electron acceptor, followed by scission of an ester to release a carboxylic acid. Three different electron acceptors were tested: nitrobenzyl, phenacyl and pyridinium. The nitrobenzyl system was ineffective, giving only photochemical decomposition and no release of the carboxylic acid. The phenacyl system also performed poorly, liberating the carboxylic acid in 20% chemical yield and 0.2% photochemical yield. The pyridinium system was most successful, and was tested for the release of two carboxylic acids: γ-amino butyric acid (GABA) and tryptophan. The caged GABA undergoes photochemical cleavage with a chemical yield of >95% and a photochemical yield of 1%; it exhibits a two-photon absorption cross section of 1100 GM at 700 nm, corresponding to a two-photon uncaging cross section of 10 ± 3 GM.

A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging.

The introduction of calcium ion (Ca(2+)) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca(2+) dynamics. However, one drawback of these indicators is that they have optimal two-photon excitation outside the near-infrared window (650-1,000 nm) where tissue is most transparent to light. To address this shortcoming, we developed a long Stokes shift RFP-based Ca(2+) indicator, REX-GECO1, with optimal two-photon excitation at <1,000 nm. REX-GECO1 fluoresces at 585 nm when excited at 480 nm or 910 nm by a one- or two-photon process, respectively. We demonstrate that REX-GECO1 can be used as either a ratiometric or intensiometric Ca(2+) indicator in organotypic hippocampal slice cultures (one- and two-photon) and the visual system of albino tadpoles (two-photon). Furthermore, we demonstrate single excitation wavelength two-colour Ca(2+) and glutamate imaging in organotypic cultures.

Photophysics and non-linear absorption of Au(I) and Pt(II) acetylide complexes of a thienyl-carbazole chromophore.

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.

Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

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.

Symmetry Breaking in Platinum Acetylide Chromophores Studied by Femtosecond Two-Photon Absorption Spectroscopy.

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 ED strength, reaching maximum value, σ2 ∼ 300 GM (1 GM = 10-50 cm4 s), for R = NPh2. At transition energies overlapping with the lowest-energy S0 → S1 transition in the one-photon absorption (1PA) spectrum, the same neutral and ED-substituted Pt complexes show weak 2PA, σ2 < 30-100 GM, which is in agreement with the nearly quadrupolar structure of these systems. Surprisingly, EW-substituted Pt complexes display a very different behavior, where the peak 2PA of the S0 → S1 transition gradually increases with increasing EW strength, reaching values σ2 = 700 GM for R = NO2, while in the S0 → Sn transition region the peak 2PEF cross section decreases. We explained this effect by breaking of inversion symmetry due to conformational distortions associated with low energy barrier for ground-state rotation of the ligands. Our findings are corroborated by theoretical calculations that show large increase of the permanent electric dipole moment change in the S0 → S1 transition when ligands with strong EW substituents are twisted by 90° relative to the planar chromophore. Our NLT results in the S0 → S1 transition region are quantitatively similar to those obtained from the 2PEF measurement. However, at higher transition energy corresponding to S0 → Sn transition region, the NLT method yields effective multiphoton absorption stronger than the 2PEF measurement in the same systems. Such enhancement is observed in all Pt complexes as well as in all ligand chromophores studied, and we tentatively attribute this effect to nearly saturated excited-state absorption (ESA), which may occur if 2PA from the ground state is immediately followed by strongly allowed 1PA to higher excited states.

Strong two-photon absorption enhancement in a unique bis-porphyrin bearing a diketopyrrolopyrrole unit.

A π-extended bis-porphyrin bridged via a diketopyrrolopyrrole unit was prepared in 5 steps. This fully conjugated π-system displays strongly distorted linear absorption, while its two-photon absorption cross-section reaches 2500-3000 GM at 940 nm. LC-like behaviour, easy orientation and low viscosity are, according to XRD, POM and DSC measurements, due to formation of plastic rather than a liquid crystal.

Alternative selection rules for one- and two-photon transitions in tribenzotetraazachlorin: quasi-centrosymmetrical π-conjugation pathway of formally non-centrosymmetrical molecule.

We compare the two-photon absorption (2PA) spectra of non-centrosymmetrical metal-free tribenzo-tetraazachlorin (H2TBTAC) and analogous symmetrical tetra-tert-butyl-phthalocyanine (H2TtBuPc). Surprisingly, despite formal lack of center of inversion, the 2PA spectrum of H2TBTAC displays a two-photon allowed transition at 935 nm, similar to gerade-gerade (g-g) transitions observed in H2TtBuPc and in other symmetrical phthalocyanines. This transition is even better resolved in the singlet-singlet excited-state absorption spectrum. We tentatively explain the survival of the g-g transition in H2TBTAC by assuming that the main π-electron conjugation pathway in the tetraaza-substituted tetrapyrrole macrocycle bypasses the outer parts of the two oppositely located isoindole rings and thus renders the optically responsive core of the chromophore quasi-centrosymmetrical. By using the independently measured ground- and excited-state absorption extinction coefficients, we also show that the two-photon absorptivity can be quantitatively explained by a simple three-level model with the lowest energy Q1 state serving as an intermediate level.

Phenylene vinylene platinum(II) acetylides with prodigious two-photon absorption.

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).

Field demonstration of a scanning lidar and detection algorithm for spatially mapping honeybees for biological detection of land mines.

A biological detection scheme based on the natural foraging behavior of conditioned honeybees for detecting chemical vapor plumes associated with unexploded ordnance devices utilizes a scanning lidar instrument to provide spatial mapping of honeybee densities. The scanning light detection and ranging (lidar) instrument uses a frequency doubled Nd:YAG microchip laser to send out a series of pulses at a pulse repetition rate of 6.853 kHz. The scattered light is monitored to produce a discrete time series for each range. This discrete time series is then processed using an efficient algorithm that is able to isolate and identify the return signal from a honeybee in a cluttered environment, producing spatially mapped honeybee densities. Two field experiments were performed with the scanning lidar instrument that demonstrate good correlation between the honeybee density maps and the target locations.