Photophysical and Electrochemical Properties of Push-Pull Oligo(ferrocenyl-phenyleneethynylene)s: Supramolecular Orders in Molecular Films.

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) (nPE) with n = 1, 2, 3 as π-conjugated bridges. In the series, by increasing the distance between the electron-attracting and electron-donor groups, the push-pull effect decreases. The intramolecular charge transfer (D → π → A) was evaluated by static and dynamic spectroscopy, electrochemistry, and density functional theory (DFT) theoretical calculations. The longest oligomer Fc3PEBz formed the best optical quality films. A study at the atomic level by scanning tunneling microscopy (STM) revealed that the molecules self-assemble on highly ordered pyrolytic graphite (HOPG) in domains with a short-range order. Films are mesoporous and the molecules arrange in a lamellar-like pattern, with an edge-on conformation with respect to HOPG, where the conjugated backbones lie parallel to the surface. Two different assemblies were identified in the monoatomic film, which depends on the ferrocene-ferrocene or benzyl-benzyl interactions.

Hot-Band Absorption Can Mimic Entangled Two-Photon Absorption.

It has been proposed that entangled two-photon absorption (E2PA) can be observed with up to 1010 lower photon flux than its classical counterpart, therefore enabling ultralow-power two-photon fluorescence microscopy. However, there is a significant controversy regarding the magnitude of this quantum enhancement in excitation efficiency. We investigated the fluorescence signals from Rhodamine 6G and LDS798 excited with a CW laser or an entangled photon pair source at ∼1060 nm. We observed a signal that originates from hot-band absorption (HBA), which is one-photon absorption from thermally populated vibrational levels of the ground electronic state. This mechanism, which has not been previously discussed in the context of E2PA, produces a signal with a linear power dependence, as would be expected for E2PA. For the typical conditions under which E2PA measurements are performed, contributions from the HBA process could lead to a several orders of magnitude overestimate of the quantum advantage.

Witnessing the survival of time-energy entanglement through biological tissue and scattering media.

We demonstrate the preservation of the time-energy entanglement of near-IR photons through thick biological media (≤1.55 mm) and tissue (≤ 235 µm) at room temperature. Using a Franson-type interferometer, we demonstrate interferometric contrast of over 0.9 in skim milk, 2% milk, and chicken tissue. This work supports the many proposed opportunities for nonclassical light in biological imaging and analyses from sub-shot noise measurements to entanglement-enhanced fluorescence imaging, clearly indicating that the entanglement characteristics of photons can be maintained even after propagation through thick, turbid biological samples.

Two-photon absorption spectra of fluorescent isomorphic DNA base analogs.

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).

Spontaneous Symmetry Breaking Facilitates Metal-to-Ligand Charge Transfer: A Quantitative Two-Photon Absorption Study of Ferrocene-phenyleneethynylene Oligomers.

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.

Symmetry Breaking in Pyrrolo[3,2-b]pyrroles: Synthesis, Solvatofluorochromism and Two-photon Absorption.

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.

Pyrrolo[3,2-b]pyrroles-From Unprecedented Solvatofluorochromism to Two-Photon Absorption.

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.

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.

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.

Primary role of the chromophore bond length alternation in reversible photoconversion of red fluorescence proteins.

Rapid photobleaching of fluorescent proteins can limit their use in imaging applications. The underlying kinetics is multi-exponential and strongly depends on the local chromophore environment. The first, reversible, step may be attributed to a rotation around one of the two exocyclic C-C bonds bridging phenol and imidazolinone groups in the chromophore. However it is not clear how the protein environment controls this motion - either by steric hindrances or by modulating the electronic structure of the chromophore through electrostatic interactions. Here we study the first step of the photobleaching kinetics in 13 red fluorescent proteins (RFPs) with different chromophore environment and show that the associated rate strongly correlates with the bond length alternation (BLA) of the two bridge bonds. The sign of the BLA appears to determine which rotation is activated. Our results present experimental evidence for the dominance of electronic effects in the conformational dynamics of the RFP chromophore.