Ultrabright supramolecular beacons based on the self-assembly of two-photon chromophores on metal nanoparticles

Silver nanoparticles coated with a self-assembled layer of approximately 2500 chromophoric alkylthiol ligands, that exhibit a huge per particle two-photon absorption cross section (2.7 x 10-45 cm4 s photon-1) and a high fluorescence quantum yield (0.33), are reported. Polyfunctionalized variants of these nanoparticles have been produced that show reasonable solubility in water/ethanol mixtures. By virtue of the large number of tethered chromophores, these particles act as strongly two-photon absorbing nanobeacons and may have applications in fluorescence imaging and sensing.     

Metal ion sensing novel calix[4]crown fluoroionophore with a two-photon absorption property

1,3-Alternate calix[4]arene-based fluorescent chemosensors bearing two-photon absorbing chromophores have been synthesized, and their sensing behaviors toward metal ions were investigated via absorption band shifts as well as one- and two-photon fluorescence changes. Free ligands absorb the light at 461 nm and weakly emit their fluorescence at 600 nm when excited by UV-vis radiation at 461 nm, but no two-photon excited fluorescence is emitted by excitation at 780 nm. Addition of an Al(3+) or Pb(2+) ion to a solution of the ligand causes a blue-shifted absorption and enhanced fluorescence due to a declined resonance energy transfer (RET) upon excitation by one- and two-photon processes. Addition of a Pb(2+) ion to a solution of 1.K(+) results in a higher fluorescence intensity than the original 1.Pb(2+) complex regardless of one- or two-photon excitation, due to the allosteric effect induced by the complexation of K(+) with a crown loop.     

Enhanced two-photon emission in coupled metal nanoparticles induced by conjugated polymers

Interactions between noble metal (Ag and Au) nanoparticles and conjugated polymers as well as their one- and two-photon emission have been investigated. Ag and Au nanoparticles exhibited extraordinary quenching effects on the fluorescence of cationic poly(fluorinephenylene). The quenching efficiency by 37-nm Ag nanoparticles is ～19 times more efficient than that by 13-nm Au nanoparticles, and 9-10 orders of magnitude more efficient than typical small molecule dye-quencher pairs. On the other hand, the cationic conjugated polymers induce the aggregate formation and plasmonic coupling of the metal nanoparticles, as evidenced by transmission electron microscopy images and appearance of a new longitudinal plasmon band in the near-infrared region. The two-photon emissions of Ag and Au nanoparticles were found to be significantly enhanced upon addition of conjugated polymers, by a factor of 51-times and 9-times compared to the isolated nanoparticles for Ag and Au, respectively. These studies could be further extended to the applications of two-photon imaging and sensing of the analytes that can induce formation of metal nanoparticle aggregates, which have many advantages over the conventional one-photon counterparts.     

Covalent Organic Framework for Improving Near-Infrared Light Induced Fluorescence Imaging through Two-Photon Induction

Fluorescent materials exhibiting two-photon induction (TPI) are used for nonlinear optics, bioimaging, and phototherapy. Polymerizations of molecular chromophores to form π-conjugated structures were hindered by the lack of long-range ordering in the structure and strong π–π stacking between the chromophores. Reported here is the rational design of a benzothiadiazole-based covalent organic framework (COF) for promoting TPI and obtaining efficient two-photon induced fluorescence emissions. Characterization and spectroscopic data revealed that the enhancement in TPI performance is attributed to the donor-π-acceptor-π-donor configuration and regular intervals of the chromophores, the large π-conjugation domain, and the long-range order of COF crystals. The crystalline structure of TPI-COF attenuates the π–π stacking interactions between the layers, and overcomes aggregation-caused emission quenching of the chromophores for improving near-infrared two-photon induced fluorescence imaging.     

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore's usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H(2)O(2)-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially "light up" in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.     

The Effect of Branching on the One- and Two-Photon Absorption,Cell Viability,and Localization of Cationic Triarylborane Chromophores with Dipolar versus Octupolar Charge Distributions for Cellular Imaging

Two different chromophores, namely a dipolar and an octupolar system, were prepared and their linear and nonlinear optical properties as well as their bioimaging capabilities were compared. Both contain triphenylamine as the donor and a triarylborane as the acceptor, the latter modified with cationic trimethylammonio groups to provide solubility in aqueous media. The octupolar system exhibits a much higher two-photon brightness, and also better cell viability and enhanced selectivity for lysosomes compared with the dipolar chromophore. Furthermore, both dyes were applied in two-photon excited fluorescence (TPEF) live-cell imaging.     

氧芴三苯胺多枝分子的双光子吸收与电化学行为

研究了3个氧芴/三苯胺衍生物: E-2,8-双(4-二苯胺基苯乙烯基)氧芴(简称OT-G1)、E-2,8-双[4-(二苯基氨基-二苯乙烯基)(4’-溴苯基)氨基-苯乙烯基]氧芴(简称OT-G1.5)和E-2,8-双-[4’,4″-二-(二苯胺基苯乙烯基)-4-二苯胺基苯乙烯基]氧芴(简称OT-G2)的双光子吸收和电化学行为. 研究结果表明, 分子“代数”从1→1.5→2增高, 氧芴三苯胺多枝分子的HOMO能级升高、双光子荧光强度和双光子吸收截面明显增大. 由于HOMO能级的升高有利于分子的电荷转移, 因而分子表现出强的双光子吸收能力, 这表明可通过电化学行为来推断出分子的双光子吸收性能.     

以N为耦合中心多枝分子的双光子上转换荧光

采用非线性透过率法研究了以N为耦合中心的多枝化合物N-[4-{2-(3,5-二-[5-(4-叔丁基苯基)-1,3,4-噁二唑-2-]苯基}-1-乙烯基}苯基}-N,N-二苯胺(BPODPA), N,N-双[4-{2-(3,5-二-[5-(4-叔丁基苯基)-1,3,4-噁二唑-2-]苯基}-1-乙烯基}苯基}-N-苯胺(BBPOPA)和N,N,N-三[4-{2-(3,5-二-[5-(4-叔丁基苯基)-1,3,4-噁二唑-2]苯基}-1-乙烯基}苯基}胺(TBPOA)的双光子吸收性质, 测定了化合物的单光子荧光光谱和双光子上转换荧光光谱, 研究了多枝化对三苯胺分子双光子吸收和双光子激发荧光性质的影响.在800 nm波长的激光激发下,化合物BPODPA、BBPOPA和TBPOA在二氯甲烷溶液中发出很强的蓝绿色双光子上转换荧光, 荧光峰分别位于502、515 和518 nm. 这些多枝结构化合物的双光子吸收截面较大, 双光子吸收增强来源于多枝分子中扩展的π共轭体系和重复单元的协同效应.     

Enhancement of two-photon absorption cross-section in macrocyclic thiophenes with cavities in the nanometer regime

The linear and nonlinear optical properties of two thiophene-based cyclic molecules have been investigated. These molecules represent nanometer sized cavities which may be useful for novel photonic devices. By virtue of long-range interactions, these chromophores serve as novel architectures for enhanced two-photon absorption (TPA) properties. Measurements of the different size ring structures showed a 550% increase in the TPA cross-section for the larger macrocycle. Electronic structure calculations have suggested an increase in coupling of the excited states in these systems as the ring size is increased. Measurements of the ultrafast transient absorption and fluorescence were carried out with these systems in order to probe the interaction between the chromophores. The results of the transient decays as well as fluorescence anisotropy decay times gives stronger proof to the suggestion of delocalized states in the cyclic macrocycles. These results provide information regarding the optical properties of these novel systems useful for potential applications in photonics.     

Sensing for intracellular thiols by water-insoluble two-photon fluorescent probe incorporating nanogel

A novel “turn-on” two-photon fluorescent probe containing a π-conjugated triarylboron luminogen and a maleimide moiety DMDP-M based on the photo-induced electron transfer (PET) mechanism for biothiol detection was designed and synthesized. By simply loading the hydrophobic DMDP-M on a cross-linked Pluronic^® F127 nanogel (CL-F127), a probing system DMDP-M/CL-F127 was established, which shows quick response, high selectivity and sensitivity to cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous phase. The DMDP-M/CL-F127 system presented the fastest response to Cys with a rate constant of 0.56 min⁻¹, and the detection limit to Cys was calculated to be as low as 0.18 μM. The DMDP-M/CL-F127 system has been successfully applied to the fluorescence imaging of biothiols in NIH/3T3 fibroblasts either with single-photon or two-photon excitation because of its high biocompatibility and cell-membrane permeability. The present work provides a general, simple and efficient strategy for the application of hydrophobic molecules to sensing biothiols in aqueous phase, and a novel sensing system for intracellular biothiols fitted for both single-photon and two-photon fluorescence imaging.     

Amino(oligo)thiophene-based environmentally sensitive biomembrane chromophores

There is a growing need for cellular imaging with fluorescent probes that emit at longer wavelengths to minimize the effects of absorption, autofluorescence, and scattering from biological tissue. In this paper a series of new environmentally sensitive hemicyanine dyes featuring amino(oligo)thiophene donors have been synthesized via aldol condensation between a 4-methylpyridinium salt and various amino(oligo)thiophene carboxaldehydes, which were, in turn, obtained from amination of bromo(oligo)thiophene carboxaldehyde. Side chains on these fluorophores impart a strong affinity for biological membranes. Compared with benzene analogues, these thiophene fluorophores show significant red shift in the absorption and emission spectra, offering compact red and near-infrared emitting fluorophores. More importantly, both the fluorescence quantum yields and the emission peaks are very sensitive to various environmental factors such as solvent polarity or viscosity, membrane potential, and membrane composition. These chromophores also exhibit strong nonlinear optical properties, including two-photon fluorescence and second harmonic generation, which are themselves environmentally sensitive. The combination of long wavelength fluorescence and nonlinear optical properties make these chromophores very suitable for applications that require sensing or imaging deep inside tissues.     

Reversal of Solvatochromism: A New Strategy to Construct Activatable Two-photon Fluorescent Probes for Sensing

Two-photon (TP) imaging with a donor-acceptor (D?A) type fluorophore is an emerging tool for bioimaging and sensing. However, current TP probes suffer from serious solvatochromic quenching in aqueous solution due to their strong intramolecular charge transfer (ICT) in excited states. In this work, based on solvatochromism reversal, we report a novel strategy to develop TP probes for bioimaging. Specifically, compared with the normal two-photon probes that showed a fluorescence off with ICT suppressed, the novel probes exhibited strong fluorescence in the aqueous solution when their ICT was inhibited. This strategy not only provides a new way for the design of high-performance TP probes, but also expands the biological analysis toolbox for use in living systems.     

Singlet oxygen generation via two-photon excited FRET

A new approach to two-photon excited photodynamic therapy has been developed. A dendritic array of eight donor chromophores capable of two-photon absorption (TPA) was covalently attached to a central porphyrin acceptor. Steady-state fluorescence measurements demonstrated that the donor chromophores transfer excited-state energy to the porphyrin with 97% efficiency. Two-photon excitation of the donor chromophores at 780 nm resulted in a dramatic increase in porphyrin fluorescence relative to a porphyrin model compound. Enhanced singlet oxygen luminescence was observed from oxygen-saturated solutions of the target compound under two-photon excitation conditions.     

细胞内铜离子的双光子荧光成像

基于无荧光的螺环结构与具有荧光的开环酰胺的平衡反应,本文合成了一个能在水基的缓冲溶液中选择性地识别Cu²⁺的罗丹明衍生物FD2.当在HEPES缓冲溶液中加入10当量的Cu²⁺时,FD2的单光子激发荧光和双光子激发荧光的强度均表现出明显的增强;更为重要的是,运用双光子荧光显微技术可以选择性地对活细胞内Cu²⁺进行成像.     

Nano-oncology: drug delivery, imaging, and sensing

Innovation in the last decade has endowed nanotechnology with an assortment of tools for delivery, imaging, and sensing in cancer research—stealthy nanoparticle vectors circulating in vivo, assembled with exquisite molecular control, capable of selective tumor targeting and potent delivery of therapeutics; intense and photostable quantum dot-based tumor imaging, enabling multicolor detection of cell receptors with a single optical excitation source; arrays of semiconducting nanowire and carbon nanotube sensor elements for selective multiplexed sensing of cancer markers without the need for probe labeling. These rapidly emerging tools are indicative of a burgeoning field ready to expand into medical applications. This review attempts to outline most of the current nanoparticle toolset for therapeutic release by liposomes, dendrimers, smart polymers, and virus-based systems. Advantages of nanoparticle-based imaging and targeting by use of nanoshells and quantum dots are also explored. Finally, emerging nanoelectronics-based sensing and a global discussion on the utility of each nanoparticle system addresses their fundamental advantages and shortcomings in cancer research.     

Synthesis and Characterization of Dipolar Two-Photon Absorption Chromophores

It is a new interest to explore novel organic two-photon absorption (TPA) chromophores with large TPA cross section for their potential applications in various fields, such as three-dimensional optical data storage, micro- and nano-fabrication, biological imaging and optical limiting, and so on. As far as asymmetrical D-π-A type chromophores were concerned, larger TPA cross section can be obtained with the enhancement of electron-donation/electron-acception ability.[1] Recently, we have synthesized a series of new D-π-A type compounds. In these chromophores, stilbene or phenyl cyclo-bridged hexatriene is served as π-bridge, which terminated with a number of amino groups and dicyanomethylene unit (Scheme 1). All the chromophores can be obtained by the condensation reaction of aldehydes and active methyl or methenyl groups in good yields (72%～81%). The chromophores have been characterized by 1H NMR,IR, UV-vis, PL and EA. All these five chromophores in dilute solutions show strong red fluorescence (Table 1). The studies on the TPA properties are in progress.     

Two-photon absorption in aqueous micellar solutions

Addition of the surfactant sodium dodecyl sulfate at concentrations above the critical micelle concentration increases the fluorescence quantum yield and the two-photon absorption cross-section of charged [2.2]paracyclophane chromophores containing pairs of D-pi-D chromophores. The resulting spectra in the micellar solutions are very similar to those obtained for neutral isostructural analogues in toluene. The measured etadelta values are 1300 GM for 1C and 1920 GM for 2C, which are comparable or larger to those in toluene. These results highlight possible misleading interpretation of two-photon-induced emission for evaluating the concentration of labeled substrates used in two-photon microscopy and provide guidelines for designing molecular structures with optimized two-photon action cross-sections in water.     

双光子荧光探针研究及其应用

双光子荧光显微成像兼具诸如近红外激发、暗场成像、避免荧光漂白和光致毒、定靶激发、高横向分辨率与纵向分辨率、降低生物组织吸光系数及降低组织自发荧光干扰等特点而显著地优于单光子荧光显微成像,为生命科学研究提供了更为锐利的工具。而用于像离子的含量及其对生理的影响、离子参与的生理活动机制、离子与分子的作用、特定分子的分布及其相互作用等方面研究的双光子荧光探针,是实现成像的关键。双光子荧光探针的研究旨在促进双光子荧光显微镜应用的发展,促进生命科学、医学科学的快速发展,同时也带动双光子荧光探针所隶属的化学这一学科的发展。因此对双光子荧光探针的研究具有重要的理论和实践意义。该文综述了双光子荧光显微成像的优点、双光子荧光探针设计的原理及双光子荧光探针在离子分析方面的应用,并展望了这类荧光探针的发展趋势与应用前景。     

Novel ruthenium(II) and zinc(II) complexes for two-photon absorption related applications

Two new fluorene derivatized 1,10-phenanthroline ligands and related tris-chelate Ru(II) or Zn(II) coordination complexes have been synthesised. The linear and nonlinear (two-photon induced fluorescence) photophysical measurements have contributed to highlight the possibility to tune the absorption spectral range and excited lifetime, depending on ligand substitution and nature of the metal. More significantly, the observation of two-photon absorption (TPA) associated with long-lived metal-to-ligand charge-transfer (MLCT) excited states in the Ru(II)-based chromophores, opens a wide range of applications in the near infrared.     

多枝[1,3,4]-噁二唑衍生物的双光子吸收和光功率限幅特性

采用非线性透过率法测定了多枝[1,3,4]-噁二唑衍生物的双光子吸收性质. 测定了化合物的单光子荧光光谱和双光子荧光光谱, 在800 nm波长的激光激发下, 9-乙基-3,6-双{5-(4-叔丁基苯基)-[1,3,4] 噁二唑-2-苯乙烯基}-咔唑(3)和三-{5-(4-叔丁基苯基)-[1,3,4] 噁二唑-2-苯乙烯基-4-苯基}-胺(4)能够发出很强的蓝色和黄绿色双光子上转换荧光, 荧光峰分别位于485和547 nm. 这些多枝结构化合物的双光子吸收截面较大(数值超过104 GM), 并具有很强的光限幅效应. 多枝分子中重复单元的推拉电子结构和协同效应有效地增强了分子的双光子吸收性质.