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 S-0 -> S-1 band, while the trans complexes show quadrupolar behavior with a forbidden 0-0 transition. In the S-0( )-> S-n 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 (S-1 -> S-n). 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 pi-conjugated chromophore.

AbstractWe 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, 0–107 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.

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.

The majority of protein functions are governed by their internal local electrostatics. Quantitative information about these interactions can shed light on how proteins work and allow for improving/altering their performance. Green fluorescent protein (GFP) and its mutation variants provide unique optical windows for interrogation of internal electric fields, thanks to the intrinsic fluorophore group formed inside them. Here we use an all-optical method, based on the independent measurements of transition frequency and one- and two-photon absorption cross sections in a number of GFP mutants to evaluate these internal electric fields. Two physical models based on the quadratic Stark effect, either with or without taking into account structural (bond-length) changes of the chromophore in varying field, allow us to separately evaluate the long-range and the total effective (short- and long-range) fields. Both types of the field quantitatively agree with the results of independent molecular dynamic simulations, justifying our method of measurement.

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 modular approach to photo-labile protecting groups based on photoinduced electron transfer, providing high sensitivity to two-photon excitation.

Abstract A fiber sensor array for subsurface CO 2 concentration measurements was developed for monitoring geologic carbon sequestration sites. The fiber sensor array uses a single temperature-tunable distributed feedback (DFB) laser operating with a nominal wavelength of 2.004 μm. Light from this DFB laser is directed to one of the four probes via an inline 1 × 4 fiber optic switch. Each of the four probes is buried and allows the subsurface CO 2 to enter the probe through Millipore filters that allow the soil gas to enter the probe but keeps out the soil and water. Light from the DFB laser interacts with the CO 2 before it is directed back through the inline fiber optic switch. The DFB laser is tuned across two CO 2 absorption features, where a transmission measurement is made allowing the CO 2 concentration to be retrieved. The fiber optic switch then directs the light to the next probe where this process is repeated, allowing subsurface CO 2 concentration measurements at each of the probes to be made as a function of time. The fiber sensor array was deployed for 58 days beginning from June 19, 2012 at the Zero Emission Research Technology field site, where subsurface CO 2 concentrations were monitored. Background measurements indicate that the fiber sensor array can monitor background levels as low as 1000 parts per million (ppm). A 34-day subsurface release of 0.15 tones CO 2 / day began on July 10, 2012. The elevated subsurface CO 2 concentration was easily detected by each of the four probes with values ranging over 60,000 ppm, a factor of greater than 6 higher than background measurements.

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.

Two-photon (2P) microscopy is the preferred method for imaging fluorescent proteins and biosensors in living, thick tissues. Over the past decade, many groups have worked to improve single photon (1P) properties of fluorescent proteins, but little has been done to improve their 2P properties. This is important because 2P properties can be quite different from the 1P properties. Our goal was to create a system for screening libraries of randomly mutagenized fluorescent proteins for better 2P properties. We created an optical system for collecting 1- and 2-photon induced fluorescence images from entire petri dishes of E. coli colonies. Libraries of randomly mutagenized eGFP were screened for improved 2P/1P fluorescence ratios. Screening the first round of evolution showed a slightly larger ratio spread outside the range of parent eGFP. One hundred of these clones were subjected to a second round of screening. Colonies that showed reproducible 2P/1P ratios greater than the parent eGFP were selected to move forward. A selective pool of the best 18 clones (library A), and a more inclusive pool of the best 59 clones (library B), were used for a new round of gene shuffling and random mutagenesis. After only two rounds of evolution, libraries A and B contained at least 5 distinct mutant populations with significantly increased 2P/1P ratios. Sequence analysis of 96 clones from these libraries revealed that every clone harbored at least one mutation. Eleven unique mutants were used for a third round of evolution. Screening and sequence analysis of the third library, reveals thirteen mutations that either individually (V68M, S72G, V163A, T203I) or in combination (E6G, T65S, Q80R, N105S, D117G, N121S, Q184R, S202N, V219I) significantly shift the 2P/1P fluorescence ratios.

Two-photon laser scanning microscopy (TPLSM) has several advantages over one-photon confocal microscopy, including deeper tissue penetration, higher signal-to-background ratio, and less photodamage in the out-of-focus volume. However, due to very high instantaneous light intensities in the focal volume, the probability of further, stepwise resonant photon(s) absorption increases dramatically, leading to very efficient bleaching of a probe. To deal with this challenge one has to understand the underlying mechanisms. Here we measured the power dependence of multiphoton bleaching rates of several red fluorescent proteins expressed in live E. coli cells under two-photon microscope conditions. To clarify the photophysical mechanisms, we also used much lower repetition rate (1 kHz) and different pulse durations in experiments in vitro with Ti:Sa amplifier excitation. Our experimental data supported by quantum mechanical calculations of the chromophore in protein environment are consistent with the mechanism of ultrafast (<150 fs) singlet-singlet stepwise absorption of one or two additional photons following initial simultaneous two-photon absorption. In the DsRed2 protein, the third photon absorption most probably results in an ultrafast electron transfer (ET) from the anionic chromophore to an excited Rydberg state of a nearby positive amino acid residue (e.g. K163+). The transient radical state of the chromophore tends to accept an electron from the deprotonated E215- amino acid, thus promoting the first step of the recently established decarboxylation reaction. In mFruits proteins, the third photon promotes an ET from the chromophore to the 4S Rydberg state of nearby K70+ residue with its subsequent photoionization by the fourth photon.

The introduction of calcium ion (Ca2+) indicators based on red fluorescent proteins (RFPs) has created new opportunities for multicolour visualization of intracellular Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ and glutamate imaging in organotypic cultures. Current calcium-sensitive probes based on red fluorescent proteins are unsuitable for two-photon excitation at the near-infrared wavelengths commonly used for green fluorescent probes. Wu et al. use a structure-guided approach to engineer a red fluorescent probe with optimal two-photon excitation at these wavelengths.

Dyes exhibiting efficient two-photon absorption (2PA) are in demand for a wide range of applications, and meso–meso butadiyne-linked porphyrin dimers (bis-porphyrins) are promising chromophores in this area.

Two-photon excitation of fluorescent proteins (FPs) is widely used in imaging whole organisms or living tissues. Many different FPs are now available but these proteins have only been optimized for their one-photon properties. We have developed a technique for screening entire libraries of E. coli colonies expressing FPs that utilizes multiple wavelengths of linear excitation as well as two-photon excitation. Single mutations in a particular protein that affect one or two-photon properties are easily identified, providing new views of structure/function relationships. An amplified femtosecond Ti: sapphire laser and a spectrally filtered lamp source are used to acquire the fluorescence signals of up to similar to 1000 E. coli colonies on a standard Petri dish. Automation of the analysis and acquisition of the fluorescent signals makes it feasible to rapidly screen tens of thousands of colonies. In a proof of principle experiment with the commonly used EGFP, we used two rounds of error prone PCR and selection to evolve new proteins with shifted absorption and increased two-photon cross sections at 790nm. This method of screening, coupled with careful measurements of photo bleaching dynamics and two-photon cross sections, should make it possible to optimize a wide variety of fluorescent proteins and biosensors for use in two-photon microscopes.

Variation of two-photon absorption (2PA) of organic chromophores as a function of the environment such as different solvent polarity has important implications for practical applications of nonlinear optical (NLO) materials but could be also used as a sensitive probe of local inter- and intra-molecular interactions. We report measurement of the 2PA spectrum in the broad wavelength range 700-1000 nm of a benchmark NLO chromophore 4-Dimethylamino-4’-nitrostilbene (DANS) in a series of solvents with varying polarity by using a femtosecond nonlinear-optical transmission method with a nearly-collimated laser beam. The 2PA peak of DANS shifts systematically from 834 nm in a low dielectric constant solvent isobutyl isobutyrate (dielectric constant = 4) to 892 nm in a highly polar solvent DMSO (dielectric constant = 48), which quantitatively follows the corresponding solvatochromic shift of the S0→S1 transition peak in the linear absorption spectrum, and indicates that DANS has a significant ground state permanent electric dipole moment that interacts with the solvent environment. The corresponding change of the S0→S1 transition peak 2PA cross section in solvents with different polarity is quite large, ranging from 160 GM in isobutyl acetate to 240 GM in 1,3-Dioxolane. However, this variation exhibits no direct correlation with the solvent polarity. This effect may be tentatively attributed to the change of the excited state permanent electric dipole moment due to the instantaneous S0→S1 transition. Our results confirm that accurate measurement of 2PA spectra may serve as a probe of local molecular-level interactions.

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.

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 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.

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.