One-photon Research Articles

Dual-Mode Gold Nanocluster-Based Nanoprobe Platform for Two-Photon Fluorescence Imaging and Fluorescence Lifetime Imaging of Intracellular Endogenous miRNA.

Bioimaging is widely used in various fields of modern medicine. Fluorescence imaging has the advantages of high sensitivity, high selectivity, noninvasiveness, in situ imaging, and so on. However, one-photon (OP) fluorescence imaging has problems, such as low tissue penetration depth and low spatiotemporal resolution. These disadvantages can be solved by two-photon (TP) fluorescence imaging. However, TP imaging still uses fluorescence intensity as a signal. The complexity of organisms will inevitably affect the change of fluorescence intensity, cause false-positive signals, and affect the accuracy of the results obtained. Fluorescence lifetime imaging (FLIM) is different from other kinds of fluorescence imaging, which is an intrinsic property of the material and independent of the material concentration and fluorescence intensity. FLIM can effectively avoid the fluctuation of TP imaging based on fluorescence intensity and the interference of autofluorescence. Therefore, based on silica-coated gold nanoclusters (AuNCs@SiO2) combined with nucleic acid probes, the dual-mode nanoprobe platform was constructed for TP and FLIM imaging of intracellular endogenous miRNA-21 for the first time. First, the dual-mode nanoprobe used a dual fluorescence quencher of BHQ2 and graphene oxide (GO), which has a high signal-to-noise ratio and anti-interference. Second, the dual-mode nanoprobe can detect miR-21 with high sensitivity and selectivity in vitro, with a detection limit of 0.91 nM. Finally, the dual-mode nanoprobes performed satisfactory TP fluorescence imaging (330.0 μm penetration depth) and FLIM (τave = 50.0 ns) of endogenous miR-21 in living cells and tissues. The dual-mode platforms have promising applications in miRNA-based early detection and therapy and hold much promise for improving clinical efficacy.

Two-photon photodynamic therapy based on FRET using tumor-cell targeted riboflavin conjugated graphene quantum dot.

The photodynamic therapy (PDT) is considered as a noninvasive and photo-controlled treatment for various cancers. However, its potential is not fully developed as current clinically approved photosensitizers (PSs) mainly absorb the light in the UV-visible region (less than 700nm), where the depth of penetration is inadequate for reaching tumor cells under deeper tissue layers. Furthermore, the lack of specific accumulation capability of the conventional PSs in the tumor cells may cause serious toxicity and low treatment efficiency. To address these problems, riboflavin (Rf) conjugated and amine-functionalized nitrogen-doped graphene quantum dots (am-N-GQD) are herein proposed. Rf functions as both photosensitizer and targeting ligand by indirect excitation through intra-particle fluorescence resonance energy transfer (FRET) via two-photon (TP) excited am-N-GQD, to enhance the treatment depth, and further am-N-GQD-Rf accumulation in cancer cells using Rf transporter family (RFVTs) and Rf carrier proteins (RCPs). The one-photon (OP) and two-photon(TP)-PDT effect and cellular internalization ability of the am-N-GQD-Rf were investigated in vitro in different cancel cell lines. Besides the excellent cellular uptake as well TP-PDT capability, the superior biocompatibility of am-N-GQD-Rf in vitro makes it promising candidate in PDT.

EXACT CALCULATIONS FOR ONE PHOTON MICROMASER WITH COHERENT INPUTS I

We report a numerical simulation of the action of the one 85 Rb atom micromaser which shows that the micromaser field evolves towards a steady state equilibrium when the condition that Tp t int satisfies. In this steady state regime the cavity field reaches a steady state after a sufficient number of atoms have passed through the cavity. The cavity temperature T and the cavity quality factor Q can produce a quantitive change when one of them or the two are changed but they are not responsible for the qualitative changes in the micromaser field. Evidently the only responsible parameter for these qualitative changes is the repetition time T p namely if T p t int then the micromaser field evolution is towards a steady state equilibrium. In contrast when T p reduced to the order of t int then the micromaser field evolution is controlled by the trapping state dynamics – no steady state is apparent in this case. The first going-up trapping state at n=3 plays a significant role in the early dynamics of the micromaser field. Moreover the state of the field ultimately evolves towards a mixed state rather than a pure state.

Two-Photon CQDs-Based Dual-Mode Nanoprobe for Fluorescence Imaging and Magnetic Resonance Imaging of Intracellular Wide pH.

Biological fluorescence imaging technologies have attracted a lot of attention and have been widely used in biomedical fields. Compared with other technologies, fluorescence imaging has a lower cost, higher sensitivity, and easier operation. However, due to the disadvantages of one-photon (OP) fluorescence imaging, such as low spatial and poor temporal resolution and poor tissue permeability depth, the application of OP fluorescence imaging has some limitations. Though two-photon (TP) fluorescence imaging can well overcome these shortcomings of OP, the single-mode imaging remains deficient. Therefore, dual-mode imaging combined with TP imaging and magnetic resonance imaging (MRI) can make up for the deficiency well, which make dual-mode imaging for the early diagnosis of diseases more accurate. Hence, a dual-mode nanoprobe TP-CQDs@MnO2 was designed for probing the fluorescence/MR dual-mode imaging strategy of intracellular H+ by using TP-CQDs (two photon-carbon quantum dots) and MnO2 nanosheets. The MnO2 nanosheets treated as fluorescence quenching agents of TP-CQDs exhibited a supersensitive response to H+, which made the fluorescence signals turn "off" to "on" for TP fluorescence imaging, in the meantime, large amounts of Mn2+ were generated for MRI. A dual-mode nanoprobe TP-CQDs@MnO2 can monitor intracellular wide pH (4.0-8.0), and the fluorescence intensity of TP-CQDs@MnO2 has recovered up to more than six times and the corresponding results of MRI were satisfactory. TP fluorescence imaging of cells and tissues showed higher detection sensitivity and deeper tissue penetration (240.0 μm) than OP. The dual-mode imaging platform hold great promise for pH-related early diagnosis and treatment, which has great potential to improve clinical efficacy.

Photochemical generation of the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical from caged nitroxides by near-infrared two-photon irradiation and its cytocidal effect on lung cancer cells.

Novel caged nitroxides (nitroxide donors) with near-infrared two-photon (TP) responsive character, 2,2,6,6-tetramethyl-1-(1-(2-(4-nitrophenyl)benzofuran-6-yl)ethoxy)piperidine (2a) and its regioisomer 2b, were designed and synthesized. The one-photon (OP) (365 ± 10 nm) and TP (710–760 nm) triggered release (i.e., uncaging) of the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical under air atmosphere were discovered. The quantum yields for the release of the TEMPO radical were 2.5% (2a) and 0.8% (2b) in benzene at ≈1% conversion of 2, and 13.1% (2a) and 12.8% (2b) in DMSO at ≈1% conversion of 2. The TP uncaging efficiencies were determined to be 1.1 GM at 740 nm for 2a and 0.22 GM at 730 nm for 2b in benzene. The cytocidal effect of compound 2a on lung cancer cells under photolysis conditions was also assessed to test the efficacy as anticancer agents. In a medium containing 100 μg mL−1 of 2a exposed to light, the number of living cells decreased significantly compared to the unexposed counterparts (65.8% vs 85.5%).

Photoelectron Transfer at ZnTPyP Self-Assembly/TiO2 Interfaces for Enhanced Two-Photon Photodynamic Therapy

Two-photon (TP) absorption nanomaterials are highly desirable for deep-tissue clinical diagnostics and orthotopic disease treatment. Here, a well-designed core/shell nanostructure was successfully synthesized with a ZnTPyP self-assembly nanocrystal (ZSN) inner core coated by a homogeneous TiO2 layer outside (ZSN-TO). The ZSN is a good photosemiconductor, showing both one-photon (OP) and TP absorption properties for red fluorescence emission and electron-hole pair generation; TiO2 with good biocompatibility acts as the electron acceptor, which can transfer photoelectron from ZSN to TiO2 for highly effective electron-hole separation, favoring the production of long-life superoxide anion (O2•-) by electrons and oxygen and strong oxidizing hydroxyl radical (•OH) by holes and surrounding H2O. Once pretreated with ZSN-TO, the simultaneous OP-405 nm or TP-800 nm laser stimulation and fluorescent imaging of reactive oxygen species (ROS) showed dynamical and continuous generation of ROS in HeLa cells, with cytotoxicity significantly increasing via the type-1-like photodynamic therapy process. The results demonstrated that the combination of organic ZSN with inorganic TiO2 has great applications as an excellent photosensitizer for deep-tissue fluorescent imaging and noninvasive disease treatment via TP photodynamic therapy.

Ligand-displacement-based two-photon fluorogenic probe for visualizing mercapto biomolecules in live cells, Drosophila brains and zebrafish.

Investigating the change in expression level of mercapto biomolecules (GSH/Cys/Hcy) necessitates a rapid detection method for a series of physiological and pathological processes. Herein, we present a ligand-displacement-based two-photon fluorogenic probe based on an Fe(iii) complex, TPFeS, which is a GSH/Cys/Hcy rapid detection fluorogenic probe for in vitro analysis and live cell/tissue/in vivo imaging. The "in situ" probe is non-fluorescent and was prepared from a 1 : 2 ratio of Fe(iii) and TPS, a novel two-photon (TP) fluorophore with excellent one-photon (OP) and TP properties under physiological conditions, as a fluorescent ligand. This probe shows a rapid and remarkable fluorescence restoration (OFF-ON) property due to the ligand-displacement reaction of mercapto biomolecules in a recyclable manner in vitro. A significant two-photon action cross-section, good selectivity for biothiols, low cytotoxicity, and insensitivity to pH over the biologically relevant pH range allowed the direct visualization of mercapto biomolecules at different levels between normal/drug-treated live cells, as well as in Drosophila brain tissues/zebrafish based on the use of two-photon fluorescence microscopy.

Design and Synthesis of a Caged Carboxylic Acid with a Donor-π-Donor Coumarin Structure: One-photon and Two-photon Uncaging Reactions Using Visible and Near-Infrared Lights.

A caged carboxylic acid with a novel two-photon (TP)-responsive donor-π-donor coumarin backbone with a quadrupolar nature was designed and synthesized in this study. The newly synthesized coumarin derivative showed a strong one-photon (OP) absorption band (ε ≈ 29000 cm-1 M-1) in the visible region (>∼400 nm). Time-dependent density functional theory calculations predicted a sizable TP absorption cross-section with a maximum at ∼650 nm significantly lager than that related to the OP absorption band. This is confirmed experimentally using TP excited fluorescence in the fs regime that leads to TP absorption cross-section of 18 and 5.6 GM at 680 and 760 nm, respectively. The OP photolysis (400 nm) and near-infrared-TP photolysis (750 nm) of the caged benzoic acid resulted in a clean formation of benzoic acid and an aldehyde.

A series of terpyridine-based zinc(ii) complexes assembled for third-order nonlinear optical responses in the near-infrared region and recognizing lipid membranes.

Two-photon (TP) microscopy has advantages for biological imaging in that it allows deeper tissue-penetration and excellent resolution compared with one-photon (OP) microscopy. Herein, two-photon activated nonlinear optical (NLO) materials, a terpyridine derivative ligand with a donor-acceptor (D-A) model and its corresponding four zinc(ii) complexes (Z1-Z4), were assembled and fully characterized. Based on their systematic photophysical investigations, it was found that the third-order NLO response in the near-infrared (NIR) region was significantly enhanced for the symmetrical complex Z4 compared with the other asymmetric complexes (Z1-Z3). To further explore its utility in biological systems, Z4 was selected as a two-photon probe for cellular lipid membranes, which was confirmed both by two-photon confocal microscopy and transmission electron microscopy (TEM). Furthermore, it was found that the fluorescence intensity was enhanced for complex Z4 with the addition of bioactive liposomes (1 μg mL-1). Its recognition mechanism was demonstrated by experimental methods, as well as molecular modeling calculations. These findings should open a new pathway for functional metal complexes as lipid membrane targeting probes.

Symmetry-Dependent Intramolecular Charge Transfer Dynamics of Pyrene Derivatives Investigated by Two-Photon Excitation.

Intramolecular charge transfer (ICT) processes in two-photon (TP) allowed states were investigated using three pyrene derivatives N1, N2C, and N2T, which have different molecular symmetry depending on the number and position of N,N-dimethylaniline donating substituents. On the basis of steady-state and nondegenerate two-photon absorption measurements, we investigated femtosecond transient absorption (TA) spectra by one-photon (OPE) and two-photon excitation (TPE). In the analysis of TA spectra, we discovered that the transfer rate from locally excited state to the ICT state by TPE is slower than that by OPE, indicating that the energy barrier between the TP and ICT states is higher than that between the one-photon (OP) allowed and ICT states. Furthermore, we demonstrated that ICT dynamics in a TP state are affected by molecular symmetry through the disappearance of stimulated emission from the locally excited state in the TA spectra of N2T obtained by TPE. We believe that our findings will provide fundamental information for a better understanding of excited-state ICT dynamics.

A unique iridium(III) complex-based chemosensor for multi-signal detection and multi-channel imaging of hypochlorous acid in liver injury

Although hypochlorous acid (HOCl) has long been associated with a number of inflammatory diseases in mammalian bodies, the functions of HOCl in specific organs at abnormal conditions, such as liver injury, remain unclear due to its high reactivity and the lack of effective methods for its detection. Herein, a unique Ir(III) complex-based chemosensor, Ir-Fc, was developed for highly sensitive and selective detection of HOCl. Ir-Fc was designed by incorporating a ferrocene (Fc) quencher to a Ir(III) complex through a HOCl-responsive linker. In the presence of HOCl, the fast cleavage of Fc moiety in less than 1s led to the enhancement of photoluminescence (PL) and electrochemical luminescence (ECL), by which the concentration of HOCl was determined by both PL and ECL analysis. Taking advantages of excellent properties of Ir(III) complexes, optical and electrochemical analyses of the response of Ir-Fc towards HOCl were fully investigated. Followed by the measurements of low cytotoxicity of Ir-Fc by MTT analysis, one-photon (OP), two-photon (TP) and lifetime imaging experiments were conducted to visualise the generation of HOCl in live microphage and HepG2 cells, and in zebrafish and mouse, respectively. Furthermore, the generation and distribution of HOCl in liver cells and liver injury of zebrafish and mouse were investigated. The results demonstrated the applicability of Ir-Fc as an effective chemosensor for imaging of HOCl generation in mitochondria of cells and liver injury in vivo, implying the potential of Ir-Fc for biomedical diagnosis and monitoring applications.

Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

A two-photon probe for Al3+ in aqueous solution and its application in bioimaging

A salicylimine probe L with a simple structure has been researched more in-depth on fluorescence sensor properties based on two-photon (TP) absorption. L displays excellent selective turn-on fluorescence response for Al3+ in hexamethylenetetramine-buffered (HMTA) aqueous solution (0.3M, pH=5.8) under one-photon (OP) excitation. With the help of OP fluorescence, TP fluorescence titration, UV-spectra titration and Job's plot, the stoichiometric ratio of L with Al3+ was determined to be 1:1. The coordination sites and the coordination mechanism of L with Al3+ were analyzed in detail through 1H NMR data. Not only with a detection limit of 5.2×10−9M in vitro, but also the probe has been successfully used in the live cells and tissues for the imaging of Al3+ with TP fluorescence microscopy due to the enlarged TP cross section, providing a novel testing method for measuring Al3+ in solution or cell tissue with low autofluorescence and cytotoxicity.

Lanthanide homometallic and d–f heterometallic MOFs from the same tripodal ligand: structural comparison, one photon (OP) vs. two photon (TP) luminescence and selective guest adsorption behavior

In this paper, we report the design of a new functionalized tripodal ligand triCB-NTB (4,4′,4′′-(2,2′,2′′-nitrilotris(methylene)tris(1H-benzo[d]imidazole-2,1-diyl)tris(methylene))tribenzoic acid) and its assembly of Eu(III) homometallic or Zn(II)-Eu(III) heterometallic lanthanide-based metal–organic frameworks (MOFs). C3 symmetric Eu(III) center is achieved in the homometallic MOF, [Eu(triCB-NTB)]·DMAc·4H2O (1). The C3 axis of the three coordinated imino N atoms on the propeller-extended central triCB-NTB ligand coincides with the C3 axis that directs the three para-methyl carboxylic benzene arms on three surrounding ligands coordinating with the central Eu(III) simultaneously. Therefore, a (6, 3) pattern is formed in the framework with relatively large porosity. In the Zn-Eu heterometallic framework, [EuZn(triCB-NTB)(H2O)(Cl)2]·2DMAc·H2O (2), the N atoms on the triCB-NTB are coordinated with Zn(II) and the carboxylic O atoms are coordinated with Eu(III). A paddle-wheel linked Eu(III)2 cluster is formed and 1D loop-and-chain structure is obtained. Two ligands, two Zn(II) ions and two Eu(III)2 clusters constitute one box-like unit on the loop chain. Due to the non-inversion symmetry imposed on the central Eu(III) and the large polarizability of the ligand, both one photon (OP) luminescence based on the energy transfer from the ligand to Eu3+ and two photon (TP) luminescence based on the hypersensitive transition of the Eu3+ ions are observed in the two lanthanide-based MOFs. This satisfies the urgent needs of multi-mode luminescent lanthanide complex. Simultaneously, N2 gas and selective vapor adsorptions were also detected in the porous structure of complexes 1 and 2 due to their benzene-filled channels. The six-pointed-star like cavities in 1 can let the gas and vapor molecules go in and out smoothly subject to changes in the pressure, while the narrower and more irregular channels in 2 show irreversible adsorption behavior for benzene and cyclohexane vapor molecules in vacuum under room temperature.

Sodium‐Ion‐Selective Two‐Photon Fluorescent Probe for In Vivo Imaging

Sodium ion plays critical roles in many physiological and pathological processes such as nerve conduction, muscle and heart contraction; it modulates the electrolyte level, cation transport, and cell volume. [1] To understand these functions, a number of one-photon (OP) fluorescent probes have been developed, with SBFI and Sodium Green (SG) being commercially available (Figure 1). [2] A disadvantage of these probes is that they are bulky and suffer from difficulties in loading into live cells. The second problem, which is common for most OP probes, is their short excitation wavelength ( 500 μm), localized excitation, and prolonged observation time. [3] Using TPM, one can image intact tissue for a long period of time with minimum interference from tissue preparation artifacts that can extend more than 70 μm into the tissue slice. However, there is no report on the TP probe for Na+ that is capable of imaging [Na + ]i deep inside living tissues. To address both of these problems, we designed a two photon (TP) probe (ANa1) derived from 2acetyl-6-(dimethylamino)naphthalene (acedan) as the TP fluorophore and 1,7-diaza-15-crown-5 as the Na + ion receptor by considering the following requirements (Figure 1); (i) small MW for cell permeability; (ii) high selectivity for Na + ions; (iii) significant TP cross section for bright TPM image; (iv) large spectral shifts in different environments for discrimination between the cytosolic and membrane-bound probes; and (v) high photostability. Herein, we report that ANa1 is capable of detecting intracellular free Na + ions in live cells and tissues at >100 μm depth for a long period of

Two‐Photon Absorption of Bis[4‐(N,N‐diphenylamino)phenylethynyl]arenes

Attractive chromophores for photoscience: Two-photon (TP) absorption (TPA) of ethynyl chromophores 2–5 (see picture) was studied using femtosecond excitation between 710–950 nm. Compounds 2–5 possess higher excitation energies for the TP-excited than for the one-photon (OP) excited states. Chromophore 2 exhibits an additional slight absorption band at the S0→S1 transition. This is explained by a vibronic coupling mechanism.

Two‐photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP

Summary Two-photon (TP) excitation (820-1150 nm) and emission (280-700 nm) spectra for the fluorescent proteins (FPs) ECFP3, EGFP3 and EYFP3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one-photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

One‐ and Two‐Photon Photochemistry and Photophysics of Poly(arylenevinylene)s Containing a Biphenyl Moiety

Photochemical and photophysical properties were investigated for poly(arylenevinylene)s containing a flexible biphenyl "hinge" unit by applying one-photon (OP) and two-photon (TP) excitation to explore excited-state properties. The poly(arylenevinylene)s were poly[(2,5-dihexyloxy-p-phenylenevinylene)-alt-(4,4'-dihexyloxy-3,3'-biphenylenevinylene)] (1), poly[(2,5-dihexyloxy-p-phenylenevinylene)-alt-(2,2'-dihexyloxy-3,3'-biphenylenevinylene)] (2), and poly[(2,5-dihexyloxy-p-phenylenevinylene)-alt-(2,2'-biphenylenevinylene)] (3). Effective emission quantum yields and related photonic properties were evaluated on a realistic per-chromophore basis using effective conjugation lengths based on the Strickler-Berg relationship. Intramolecular photocyclization was deduced to occur in the one case where the biphenyl molecular connectivity permitted the reaction, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), heteronuclear multiple-quantum coherence (HMQC)-NMR, and gel-permeation chromatography (GPC) results. The various photoprocesses could be induced by either OP or TP excitation, though the first excited singlet state is the photoactive state. The higher excitation energy of the TP excited state favors indirect population of the S, state by electronic coupling between the TP and OP excited states [lambda(max)TPE (nm): 726; delta (GM): 1=229, 2=215, 3= 109). Photochemical processes occurring from the lowest OP excited state (S1) could therefore also be indirectly induced by TP excitation.

Effect of one-photon detuning on light speed reduction in a three-level Λ-type atomic system

We have observed the reduction of group velocity of light via electromagnetically induced transparency (EIT) and measured the dependence of pulse delay on the one-photon detuning in a three-level Λ-type atomic system. The results show that due to the effect of Doppler broadening the light speed reduction is signif icant in a range of ±600MHz one-photon detuning. Under the condition of consid ering the Doppler shift of frequency, the pulse delay as a function of the one-p hoton detuning is numerically calculated. The experimental measurements are in g ood agreement with the theoretical results. These researches provide theoretical and experimental references for controlling the group velocity of light by mean s of one-photon detuning.

ChemInform Abstract: TWO‐PHOTON SPECTRUM OF PHENYLACETYLENE

The two‐photon (TP) Lb (B2)←A (A1) vapor fluorescence excitation spectrum of phenylacetylene has been obtained and analyzed. The spectrum is strongly Franck–Condon (FC) allowed (the experimentally determined value of δtotal is ∼7×10−49 cm4 s molecule−1 photon−1), with the principal FC mode being ν11 (analog of the ring breathing mode ν1 in benzene). Appreciable vibronic coupling character appears in the form of ν29 (analog of the benzene mode ν14). The TP spectrum is in striking contrast to the weakly allowed one‐photon (OP) one, which is mainly allowed by vibronic coupling and resembles that of toluene. The TP spectrum reveals such pronounced delocalization of the π electrons over the ring and acetylene moieties that strong coupling theory (akin to that appropriate for aniline) is necessary to explain the intensity arising from mixing of the charge–transfer states (acetylene→ring* and ring→acetylene*) into the final and intermediate states. Perturbation theory and INDO/S calculations suggest that the dom...