High Two-photon Absorption Cross-sections Research Articles

Two-photon absorption and spectroscopy of the lowest two-photon transition in small donor-acceptor–substituted organic molecules

We determine the dispersion of the third-order polarizability of small donor-acceptor substituted organic molecules using wavelength-dependent degenerate four-wave mixing experiments in solutions with varying concentrations. We find that donor-acceptor--substituted molecules that are characterized by extremely efficient off-resonant nonlinearities also have a correspondingly high two-photon absorption cross section. The width and shape of the first two-photon resonance for these noncentrosymmetric molecules follows what is expected from their longest wavelength absorption peak, and the observed two-photon absorption cross sections are record high when compared to the available literature data, the size of the molecule, and the fundamental limit for two-photon absorption to the lowest excited state, which is essentially determined by the number of conjugated electrons and the excited-state energies. The two-photon absorption of the smallest molecule, which only has 16 electrons in its conjugated system, is one order of magnitude larger than for the molecule called AF-50, a reference molecule for two-photon absorption [O.-K. Kim et al., Chem. Mater. 12, 284 (2000)].

Elucidating the relationship between superoxide anion levels and lifespan using an enhanced two-photon fluorescence imaging probe

We report a new reversible fluorescent two-photon (TP) probe (PY-CA) with high TP absorption cross section and pH-independent fluorescence response, which allow monitoring of O2˙(-) fluxes dynamically, selectively and sensitively. The imaging results indicate that O2˙(-) at high levels can shorten the life of Caenorhabditis elegans.

Emission turn-on and solubility turn-off in conjugated polymers: one- and two-photon-induced removal of fluorescence-quenching solubilizing groups.

The synthesis of highly efficient two-photon uncaging groups and their potential use in functional conjugated polymers for post-polymerization modification are reported. Careful structural design of the employed nitrophenethyl caging groups allows to efficiently induce bond scission by a two-photon process through a combination of exceptionally high two-photon absorption cross-sections and high reaction quantum yields. Furthermore, π-conjugated polyfluorenes are functionalized with these photocleavable side groups and it is possible to alter their emission properties and solubility behavior by simple light irradiation. Cleavage of side groups leads to a turn-on of the fluorescence while solubility of the π-conjugated materials is drastically reduced.

Diketopyrrolopyrrole-porphyrin conjugates with high two-photon absorption and singlet oxygen generation for two-photon photodynamic therapy.

Two-photon photodynamic therapy is a promising therapeutic method which requires the development of sensitizers with efficient two-photon absorption and singlet-oxygen generation. Reported here are two new diketopyrrolopyrrole-porphyrin conjugates as robust two-photon absorbing dyes with high two-photon absorption cross-sections within the therapeutic window. Furthermore, for the first time the singlet-oxygen generation efficiency of diketopyrrolopyrrole-containing systems is investigated. A preliminary study on cell culture showed efficient two-photon induced phototoxicity.

A Polyurethane-Based Positive Photoresist.

Polyurethane (PU) monomer mixtures containing commercially available o-nitrobenzyl-based photocleavable monomers have been formulated and tested as low-cost positive tone photoresists. The photolysis reaction is studied by UV spectroscopy. Well-defined micropatterns on 2 μm thick photodegradable PU films are obtained using 365 nm light exposure. This strategy is also extended to improved formulations based on synthesized o-nitrobiphenylpropyl derivatives with enhanced photochemical properties for single photon excitation and high two-photon absorption cross-sections. Improved pattern resolution in 2D and the capability of 3D resolution using a scanning laser at 780 nm is demonstrated. This work demonstrates the potential of PUs as readily available, versatile, and easy-to-use photoresist materials for low-cost lithography applications.

Two‐Photon‐Triggered Drug Delivery in Cancer Cells Using Nanoimpellers

A therapy of cancer cells: Two-photon-triggered camptothecin delivery with nanoimpellers was studied in MCF-7 breast cancer cells. A fluorophore with a high two-photon absorption cross-section was first incorporated in the nanoimpellers. Fluorescence resonance energy transfer (FRET) from the fluorophore to the azobenzene moiety was demonstrated.

Multi-photon excitation in uncaging the small molecule bioregulator nitric oxide

Multi-photon excitation allows one to use tissue transmitting near-infrared (NIR) light to access excited states with energies corresponding to single-photon excitation in the visible or ultraviolet wavelength ranges. Here, we present an overview of the application of both simultaneous and sequential multi-photon excitation in studies directed towards the photochemical delivery ('uncaging') of bioactive small molecules such as nitric oxide (NO) to physiological targets. Particular focus will be directed towards the use of dyes with high two-photon absorption cross sections and lanthanide ion-doped upconverting nanoparticles as sensitizers to facilitate the uncaging of NO using NIR excitation.

Unsaturated oxazolones as nonlinear fluorophores

Unwanted bleaching of two-photon absorption microscopy probes can be reduced by designing more efficient nonlinear fluorophores. Here the linear and nonlinear photophysical properties of unsaturated oxazolones are studied. A set of push–pull oxazolone derivatives have been designed by substituting the 2-phenyl-4-benzylidene-1,3-oxazol-5-one unit in two different positions with a combination of electron donor (dimethylaniline) and/or acceptor (nitro, nitrile and benzimidazole) groups. By engineering this green fluorescent protein analogue core we have prepared molecules with high two-photon absorption cross-sections 200–500 GM and fluorescence quantum yields up to 0.75. The factors determining the overall nonlinear emission efficiency of the new unsaturated oxazolones are discussed in terms of the nature of the substituents.

Strong Two-Photon-Induced Fluorescence from Photostable, Biocompatible Nitrogen-Doped Graphene Quantum Dots for Cellular and Deep-Tissue Imaging

Bright two-photon fluorescent probes are highly desirable to be able to optically probe biological activities deep inside living organisms with larger imaging depth, minor autofluorescence background, and less photodamage. In this study, we report the biocompatible nitrogen-doped graphene quantum dots (N-GQDs) as efficient two-photon fluorescent probes for cellular and deep-tissue imaging. The N-GQD was prepared by a facile solvothermal method using dimethylformamide as a solvent and nitrogen source. The two-photon absorption cross-section of N-GQD reaches 48,000 Göppert-Mayer units, which far surpasses that of the organic dyes and is comparable to that of the high performance semiconductor QDs, achieving the highest value ever reported for carbon-based nanomaterials. More importantly, a study of penetration depth in tissue phantom demonstrates that the N-GQD can achieve a large imaging depth of 1800 μm, significantly extending the fundamental two-photon imaging depth limit. In addition, the N-GQD is nontoxic to living cells and exhibits super photostability under repeated laser irradiation. The high two-photon absorption cross-section, large imaging depth, good biocompatibility, and extraordinary photostability render the N-GQD an attractive alternative probe for efficient two-photon imaging in biological and biomedical applications.

Functionalized Two-Photon Absorbing Diketopyrrolopyrrole-Based Fluorophores for Living Cells Fluorescent Microscopy

Two-photon excited microscopy has evolved as a routine technique for long-term cellular and in vivo imaging and is now available in most optical microscopy facilities. Classical dyes and fluorescent proteins, developed for epifluorescence or confocal microscopy, are used but unfortunately present a low efficiency upon two-photon excitation inducing the need of high excitation power (over 20 mW). To reduce this excitation power, new dyes need to be developed, allowing really low two-photon excitation power in the milliwatt or sub-milliwatt range. We report here the conception, synthesis, and physicochemical and photophysical properties of new functionalized diketopyrrolopyrrole (DPP) derivatives acting as fluorescent tags for biomolecules. They present high two-photon absorption cross-sections and bright luminescence around 600 nm. These two-photon optimized fluorophores were bioconjugated to HIV-I Tat (44-61), and their cellular localization was observed by two-photon excited microscopy using sub-milliwatt laser excitation power.

Heptametallic, Octupolar Nonlinear Optical Chromophores with Six Ferrocenyl Substituents

New complexes with six ferrocenyl (Fc) groups connected to Zn(II) or Cd(II) tris(2,2'-bipyridyl) cores are described. A thorough characterisation of their BPh4(-) salts includes two single-crystal X-ray structures, highly unusual for such species with multiple, extended substituents. Intense, visible d(Fe(II))→π* metal-to-ligand charge-transfer (MLCT) bands accompany the π→π* intraligand charge-transfer absorptions in the near UV region. Each complex shows a single, fully reversible Fe(III/II) wave when probed electrochemically. Molecular quadratic nonlinear optical (NLO) responses are determined by using hyper-Rayleigh scattering and Stark spectroscopy. The latter gives static first hyperpolarisabilities β0 reaching as high as approximately 10(-27) esu and generally increasing with π-conjugation extension. Z-scan cubic NLO measurements reveal high two-photon absorption cross-sections σ2 of up to 5400 GM in one case. DFT calculations reproduce the π-conjugation dependence of β0, and TD-DFT predicts three transitions close in energy contributing to the MLCT bands. The lowest energy transition has octupolar character, whereas the other two are degenerate and dipolar in nature.

Femto-second laser beam with a low power density achieved a two-photon photodynamic cancer therapy with quantum dots.

Focusing the femto-second (fs) laser beam on the target was the usual way to carry out a two-photon excitation (TPE) in previous photodynamic therapy (PDT) studies. However, focusing the laser deep inside the tissues of the tumor is unrealistic due to tissue scattering, so that this focusing manner seems unfit for practical TPE PDT applications. In this work, we prepared a conjugate of quantum dots (QDs) and sulfonated aluminum phthalocyanine (AlPcS) for TPE PDT, because QDs have a very high two-photon absorption cross section (TPACS) and thus QDs can be excited by an unfocused 800 nm fs laser beam with a low power density and then transfer the energy to a conjugated AlPcS via fluorescence resonance energy transfer (FRET). The FRET efficiency of the QD-AlPcS conjugate in water was as high as 90%, and the FRET process of the cellular QD-AlPcS was also observed in both KB and HeLa cells under TPE of a 800 nm fs laser. The singlet oxygen (1O2) products were produced by the QD-AlPcS under the TPE of the unfocused 800 nm fs laser via FRET mediated PDT. Moreover, the QD-AlPcS can effectively destroy these cancer cells under the irradiation of the 800 nm unfocused fs laser beam with a power density of 92 mW mm-2, and particularly the killing efficiency of the TPE is comparable to that of the commonly used one-photon excitation (OPE) at visible wavelengths. These results highlight the potential of QD-AlPcS for TPE PDT with a near infrared wavelength.

Coumarin-containing photo-responsive nanocomposites for NIR light-triggered controlled drug release via a two-photon process.

A new multifunctional nanovehicle for tumor therapy and cell imaging was fabricated by coating NIR light-responsive polymers (HAMAFA-b-DDACMM) onto the surface of octadecyltrimethoxysilane (C18)-modified hollow mesoporous silica nanoparticles (HMS@C18) via self-assembly. First, the targeting NIR light-responsive block copolymer was synthesized by the RAFT living polymerization of [7-(didodecylamino) coumarin-4-yl] methyl methacrylate with hydroxyethylacrylate and N-(3-aminopropyl) methacrylamide hydrochloride and then grafted with folic acid (FA). The copolymers could be disrupted by excitation by a femtosecond NIR light laser (800 nm) via a two-photon absorption process due to the high two-photon absorption cross-section of the coumarin moiety. In order to enhance the drug loading capacity and biological stability of the nanovehicle, HMS nanoparticles modified by hydrophobic octadecyl chains were selected as the "core", which had a considerable drug loading efficiency of more than 70%. Then the core-shell nanocomposites (HMS@C18@HAMAFA-b-DDACMM) were obtained by coating the amphiphilic copolymers onto the core via self-assembly. Under excitation by NIR light at 800 nm, the pre-loaded drugs could be released from the nanocomposites due to the degradation of the light-responsive copolymers and the release efficiency was correlated with the irradiation time and light power. The in vitro experiments indicated that the nanocomposites were easily targeted into the tumor cells that over-expressed folic acid receptor (FR(+)) such as KB cells by endocytosis. Furthermore, the copolymer itself had strong fluorescence, which could be used to track the process of drug delivery.

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).

Two-photon absorption properties of push–pull oxazolones derivatives

New fluorescent oxazolone derivatives with high two-photon absorption cross-section were synthesized. Electron donor (phenyl, dimethylaniline and furanyl) and acceptor (nitrobenzene and ethenyl-phenyl-benzimidazol) groups have been appended to the methylene end of 4-(methylidene)-2-phenyl-1,3-oxazol-5(4H)-one in order to produce an highly conjugated π-system with push–pull geometry. The linear and nonlinear optical properties of the oxazolones have been determined. The compounds with a high charge transfer from the substituent group to the oxazolone ring have relatively high two-photon absorption cross-sections (80–100 GM). The best performing nonlinear fluorophore being the benzimidazol derivative with a two-photon absorption cross-section of 80 GM and a relatively high emission quantum yield , ϕ = 0.31. ► Nonlinear optical properties of oxazolone derivatives were studied. ► Several Push–pull molecules were synthesized. ► Two-photon excited fluorescence has been observed. ► Two-photon absorption cross-sections of 80–100 GM were measured.

Octupolar Merocyanine Dyes: A New Class of Nonlinear Optical Chromophores

A set of new octupolar merocyanine chromophores was designed and synthesized. These compounds were prepared from the reaction of 1,3,5-triformyl-2,4,6-trihydroxybenzene with heterocyclic nucleophiles. Octupolar dyes were formed exclusively in their open-dye form. The one- and two-photon-absorption spectra of the dyes consist of two bands: The long-wavelength band in the two-photon absorption spectrum (a few hundreds GM above 1000 nm) matches well with the intense, long-wavelength-absorption band that is located in the visible region in the linear spectrum. Interestingly, an additional, much-more-intense TPA band in the NIR region is observed at higher energy, which corresponds to a weakly allowed one-photon electronic transition. Changing the peripheral heterocyclic moieties allows tuning of the optical properties to approach the cyanine limit (i.e., polymethine state), thus resulting in a red-shift of the low-energy one-photon-absorption band as well as to the rise of an intense two-photon-absorption band in the NIR region. To the best of our knowledge, this is the first synthesis and TPA characterization of octupolar merocyanine chromophores with typical low-bond-length alternation.

Near-infrared-emitting squaraine dyes with high 2PA cross-sections for multiphoton fluorescence imaging.

Designed to achieve high two-photon absorptivity, new near-infrared (NIR) emitting squaraine dyes, (E)-2-(1-(2-(2-methoxyethoxy)ethyl)-5-(3,4,5-trimethoxystyryl)-1H-pyrrol-2-yl)-4-(1-(2-(2-methoxyethoxy)ethyl)-5-(3,4,5-trimethoxystyryl)-2H-pyrrolium-2-ylidene)-3-oxocyclobut-1-enolate (1) and (Z)-2-(4-(dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate (2), were synthesized and characterized. Their linear photophysical properties were investigated via UV-visible absorption spectroscopy and fluorescence spectroscopy in various solvents, while their nonlinear photophysical properties were investigated using a combination of two-photon induced fluorescence and open aperture z-scan methods. Squaraine 1 exhibited a high two-photon absorption (2PA) cross-section (δ2PA), ∼20 000 GM at 800 nm, and high photostability with the photochemical decomposition quantum yield one order of magnitude lower than Cy 5, a commercially available pentamethine cyanine NIR dye. The cytotoxicity of the squaraine dyes were evaluated in HCT 116 and COS 7 cell lines to assess the potential of these probes for biomedical imaging. The viability of both cell lines was maintained above 80% at dye concentrations up to 30 μM, indicating good biocompatibility of the probes. Finally, one-photon fluorescence microscopy (1PFM) and two-photon fluorescence microscopy (2PFM) imaging was accomplished after incubation of micelle-encapsulated squaraine probes with HCT 116 and COS 7 cells, demonstrating their potential in 2PFM bioimaging.

Synthesis and Photophysical Properties of Two-Photon Absorbing Spirofluorene Derivatives

New spirofluorene-based quadrupolar two-photon absorbing dyes having triphenylamine and N,N-dibutylaniline as electron donors at the end of molcules were designed and synthesized. The third-order nonlinear optical properties of these compounds were studied using a two-photon excited fluorescence method. They were found to have high two-photon absorption cross-section owing to extended conjugation of the spirofluorene moiety. The effect of varying the donor strength could be discerned by comparing the two compounds. They were successfully used as a photosensitizers for two-photon initiated polymerization of three-dimensional micro-objects.

Structural Changes in Bacteriorhodopsin Caused by Two-Photon-Induced Photobleaching

Bacteriorhodopsin (BR) is the key protein of the halobacterial photosynthetic system. BR assembles into two-dimensional crystalline patches, the so-called purple membranes (PM), and acts as a light-driven proton pump converting light energy into the chemical energy of a proton gradient over the cell membrane. The two-photon absorption (TPA) of BR is so far not fully understood. Astonishingly high TPA cross sections have been reported, but the molecular mechanisms have not been elucidated. In this work, we address structural changes in BR and PM upon TPA, investigating its TPA photochemistry by spectroscopy, small-angle X-ray scattering, as well as electron and atomic force microscopy. We observe that TPA of BR leads to formation of an UV-absorbing N-retinyl-bacterioopsin state, which is accompanied by the loss of crystalline order in PM. FTIR and CD spectroscopy confirm that BR trimers as well as the secondary structure of the BR molecules are preserved. We demonstrate that excitation by TPA results in the photochemical reduction of the retinal Schiff base, which in turn causes a permanent asymmetric shape change of BR, similar to the one transiently observed during the photocycle-related opening and closing of the cytoplasmic proton half channel. This shape change causes PM sheets to merely roll up toward the extracellular side and causes the loss of crystallinity of PM. We present a model for the TPA photoresponse of BR, which also explains the irreversibility of the process in terms of a photochemical reduction of the Schiff base.

Upconversion fluorescent carbon nanodots enriched with nitrogen for light harvesting

Fluorescent nitrogen-enriched carbon nanodots (C-dots) of 1 to 3 nm were obtained through a one-pot reaction between melamine and glycerol. These C-dots show quantum yields up to 22% and a high two-photon absorption cross-section. The TiO2 based photoanode sensitized by these C-dots is capable of converting near IR photon energy to photocurrent.