Femtosecond Spectroscopy and Nonlinear Optical Properties of aza-BODIPY Derivatives in Solution.

The fast relaxation processes in the excited electronic states of functionalized aza-boron-dipyrromethene (aza-BODIPY) derivatives (1-4) were investigated in liquid media at room temperature, including the linear photophysical, photochemical, and nonlinear optical (NLO) properties. Optical gain was revealed for nonfluorescent derivatives 3 and 4 in the near infrared (NIR) spectral range under femtosecond excitation. The values of two-photon absorption (2PA) and excited-state absorption (ESA) cross-sections were obtained for 1-4 in dichloromethane using femtosecond Z-scans, and the role of bromine substituents in the molecular structures of 2 and 4 is discussed. The nature of the excited states involved in electronic transitions of these dyes was investigated using quantum-chemical TD-DFT calculations, and the obtained spectral parameters are in reasonable agreement with the experimental data. Significant 2PA (maxima cross-sections ∼2000 GM), and large ESA cross-sections ∼10-20  m2 of these new aza-BODIPY derivatives 1-4 along with their measured high photostability reveal their potential for photonic applications in general and optical limiting in particular.

Nature of Linear Spectral Properties and Fast Electronic Relaxations in Green Fluorescent Pyrrolo[3,4-c]Pyridine Derivative.

The electronic nature of 4-hydroxy-1H-pyrrolo[3,4-c]pyridine-1,3,6(2H,5H)-trione (HPPT) was comprehensively investigated in liquid media at room temperature using steady-state and time-resolved femtosecond transient absorption spectroscopic techniques. The analysis of the linear photophysical and photochemical parameters of HPPT, including steady-state absorption, fluorescence and excitation anisotropy spectra, along with the lifetimes of fluorescence emission and photodecomposition quantum yields, revealed the nature of its large Stokes shift, specific changes in the permanent dipole moments under electronic excitation, weak dipole transitions with partially anisotropic character, and high photostability. Transient absorption spectra of HPPT were obtained with femtosecond resolution and no characteristic solvate relaxation processes in protic (methanol) solvent were revealed. Efficient light amplification (gain) was observed in the fluorescence spectral range of HPPT, but no super-luminescence and lasing phenomena were detected. The electronic structure of HPPT was also analyzed with quantum-chemical calculations using a DFT/B3LYP method and good agreement with experimental data was shown. The development and investigation of new pyrrolo[3,4-c]pyridine derivatives are important due to their promising fluorescent properties and potential for use in physiological applications.

Protein-induced fluorescence enhancement of two-photon excitable water-soluble diketopyrrolopyrroles.

Fluorescent contrast agents are important tools in cell biology and medical imaging due to their high sensitivity and relative availability. Diketopyrrolopyrrole (DPP) derivatives have been recently studied for applications in bioimaging, but certain drawbacks due to their inherent structure have stifled progress towards their widespread implementation. Aggregation caused quenching (ACQ) associated with π-π stacking in relatively rigid extended conjugation systems as well as hydrophobicity of previously reported DPPs make most unsuitable for biological imaging applications. Addressing these deficiencies, we report the synthesis and photophysical characterization of two new water-soluble diketopyrrolopyrole (DPP) probes that exhibit pronounced protein-induced fluorescence enhancement (PIFE) upon binding serum albumin protein. In vitro studies were also performed showing low cytotoxicity for the new DPP probes. Two-photon fluorescence microscopy (2PFM) images were obtained via excitation at 810 nm and emission in the NIR window of biological transparency, illustrating the potential of these compounds as nonlinear optical bioimaging probes.

A Deoxyuridine-Based Far-Red Emitting Viscosity Sensor.

A novel deoxyuridine (dU) benzothiazolium (BZ) derivative, referred to as dU-BZ, is reported that was synthesized via Sonogashira coupling reaction methodology. The deoxyuridine building block was introduced to enhance hydrophilicity, while an alkynylated benzothiazolium dye was incorporated for long wavelength absorption to reduce potential phototoxicity that is characteristic of using UV light to excite common fluorphores, better discriminate from native autofluorescence, and potentially facilitate deep tissue imaging. An impressive 30-fold enhancement of fluorescence intensity of dU-BZ was achieved upon increasing viscosity. Fluorescence quantum yields in 99% glycerol/1% methanol (v/v) solution as a function of temperature (293-343 K), together with viscosity-dependent fluorescence lifetimes and radiative and non-radiative rate constants in glycerol/methanol solutions (ranging from 4.8 to 950 cP) were determined. Both fluorescence quantum yields and lifetimes increased with increased viscosity, consistent with results predicted by theory. This suggests that the newly-designed compound, dU-BZ, is capable of functioning as a probe of local microviscosity, an aspect examined by in vitro bioimaging experiments.

Superfluorescent squaraine with efficient two-photon absorption and high photostability.

The synthesis, linear photophysical, two-photon absorption (2PA), femtosecond transient absorption, and superfluorescence properties of a new symmetrical squaraine derivative (1) are reported. Steady-state linear spectral and photochemical properties, fluorescence lifetimes, and excitation anisotropy of 1 were investigated in various organic solvents. High fluorescence quantum yields (≈0.7) and very high photostability (photodecomposition quantum yields ≈10(-6)-10(-8)) were observed. An open-aperture Z-scan method was used to obtain 2PA spectra of 1 over a broad spectral range (maximum 2PA cross section ≈1000 GM). Excited-state absorption (ESA) and gain was observed by femtosecond transient absorption spectroscopy, in which both reached a maximum at approximately 500 fs. Squaraine 1 exhibits efficient superfluorescence. The quantum chemical study of 1 revealed the simulated vibronic nature of the 1PA and 2PA spectra were in good agreement with experimental data; this may provide the ability to predict potential advanced photonic materials.

Controlled aggregation and enhanced two-photon absorption of a water-soluble squaraine dye with a poly(acrylic acid) template.

Controlling the aggregation behavior of organic dyes is important for understanding and exploring supramolecular assembly utilizing the specific characteristics of aggregation. Regulating J-aggregation by electrostatic interactions between anionic polyelectrolytes and cationic dyes has gained growing interest. Here, we report the formation of J-aggregates of a water-soluble cationic squaraine dye, 4-(pyridinium-1-yl)butylbenzothiazolium squaraine (SQ), using poly(acrylic acid) sodium salt (PAA-Na) as a template. Electrostatic interactions between the PAA-Na polyelectrolyte and the cationic SQ dye enhanced J-aggregation; the absorbance of the resulting J-band with the polyelectrolyte template was much sharper than the absorbance of the J-aggregate formed using a high concentration of NaCl. Significantly, removal of the polyelectrolyte PPA-Na template by the introduction of calcium ions, which can form stronger ionic binding with carboxylate groups, dissociated J-aggregates, freeing the SQ molecules back to unaggregated or lower aggregate forms. To demonstrate the reversibility of the J-aggregate formation cycle, an in situ experiment was conducted that showed 60% reversibility of the second cycle. In addition, an enhancement by ca. 23 times per repeat unit of the two-photon absorption (2PA) cross section was observed at 920 nm for the polyelectrolyte template-SQ J-aggregate compared to unaggregated or lower aggregate SQ. These results suggest a prominent role of polyelectrolyte templated SQ J-aggregation in the enhancement of 2PA efficiency and provide a means of modulating supramolecular assembly.

Nature of Linear Spectral Properties and Fast Relaxations in the Excited States and Two-Photon Absorption Efficiency of 3-Thiazolyl and 3-Phenyltiazolyl Coumarin Derivatives.

Coumarin-based fluorescent agents play an important role in the manifold fundamental scientific and technological areas and need to be carefully studied. In this research, linear photophysics, photochemistry, fast vibronic relaxations, and two-photon absorption (2PA) of the coumarin derivatives, methyl 4-[2-(7-methoxy-2-oxo-chromen-3-yl)thiazol-4-yl]butanoate (1) and methyl 4-[4-[2-(7-methoxy-2-oxo-chromen-3-yl)thiazol-4-yl]phenoxy]butanoate (2), were comprehensively analyzed using stationary and time-resolved spectroscopic techniques, along with quantum-chemical calculations. The steady-state one-photon absorption, fluorescence emission, and excitation anisotropy spectra, as well as 3D fluorescence maps of 3-hetarylcoumarins 1 and 2 were obtained at room temperature in solvents of different polarities. The nature of relatively large Stokes shifts (∼4000-6000 cm-1), specific solvatochromic behavior, weak electronic π → π* transitions, and adherence to Kasha's rule were revealed. The photochemical stability of 1 and 2 was explored quantitatively, and values of photodecomposition quantum yields, on the order of ∼10-4, were determined. A femtosecond transient absorption pump-probe technique was used for the investigation of fast vibronic relaxation and excited-state absorption processes in 1 and 2, while the possibility of efficient optical gain was shown for 1 in acetonitrile. The degenerate 2PA spectra of 1 and 2 were measured by an open aperture z-scan method, and the maximum 2PA cross-sections of ∼300 GM were obtained. The electronic nature of the hetaryl coumarins was analyzed by quantum-chemical calculations using DFT/TD-DFT level of theory and was found to be in good agreement with experimental data.

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.

Two-photon absorption and time-resolved stimulated emission depletion spectroscopy of a new fluorenyl derivative.

The synthesis, comprehensive linear photophysical characterization, two-photon absorption (2PA), steady-state and time-resolved stimulated emission depletion properties of a new fluorene derivative, (E)-1-(2-(di-p-tolylamino)-9,9-diethyl-9H-fluoren-7-yl)-3-(thiophen-2-yl)prop-2-en-1-one (1), are reported. The primary linear spectral properties, including excitation anisotropy, fluorescence lifetimes, and photostability, were investigated in a number of aprotic solvents at room temperature. The degenerate 2PA spectra of 1 were obtained with open-aperture Z-scan and two-photon induced fluorescence methods, using a 1 kHz femtosecond laser system, and maximum 2PA cross-sections of ∼400-600 GM were obtained. The nature of the electronic absorption processes in 1 was investigated by DFT-based quantum chemical methods implemented in the Gaussian 09 program. The one- and two-photon stimulated emission spectra of 1 were measured over a broad spectral range using a femtosecond pump-probe-based fluorescence quenching technique, while a new methodology for time-resolved fluorescence emission spectroscopy is proposed. An effective application of 1 in fluorescence bioimaging was demonstrated by means of one- and two-photon fluorescence microscopy images of HCT 116 cells containing dye encapsulated micelles.

Two-photon absorption enhancement of polymer-templated porphyrin-based J-aggregates.

Supramolecular structures based on organized assemblies of macrocyclic chromophores, particularly porphyrin-based dyes, have attracted widespread interest as components of molecular devices with potential applications in molecular electronics, artificial light harvesting, and pharmacology. We report the formation of J-aggregates of two porphyrin-based dyes, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TSPP, 4) and an amino tris-sulfonate analogue (5) in water using a functionalized norbornene-based homopolymer, synthesized by ring-opening metathesis polymerization (ROMP). Ionic interactions of the cationic side chains (ammonium groups) of the polymer under acidic conditions with the negatively charged sulfonate groups of the porphyrins facilitated polymer template enhanced J-aggregation of the porphyrin dyes. J-Aggregation behavior was investigated photophysically by UV-vis absorption along with steady-state and time-resolved fluorescence studies. Two-photon absorption (2PA) was enhanced by about an order of magnitude for the J-aggregated TSPP relative to its free base. Significantly, the 2PA cross section of the polymer-templated TSPP J-aggregate was up to three times higher than the J-aggregated TSPP in the absence of the polymer template while the 2PA cross section for polymer-templated J-aggregates of 5 increased substantially, up to ca. 10,000 GM, suggesting a prominent role of polymer-templating to facilitate porphyrin aggregation and greatly enhance nonlinear absorption.

New Two-Photon Absorbing Squaraine Derivative with Efficient Near-Infrared Fluorescence, Superluminescence, and High Photostability.

The nature of linear photophysical and nonlinear optical properties of a new squaraine derivative 2,4-bis[4-(azetidyl)-2-hydroxyphenyl]squaraine (1) with efficient near-infrared (NIR) emission was comprehensively analyzed based on spectroscopic, photochemical, and two-photon absorption (2PA) measurements, along with quantum chemical analysis. The steady-state absorption, fluorescence, and excitation anisotropy spectra of 1 and its fluorescence emission lifetimes revealed the multiple aspects of the electronic structure of 1, including the relative orientations of the main transition dipoles, effective rotational volumes in solvents of different polarities, and a maximum molar extinction of 1.35 × 10-5 M-1·cm-1, which is unusually small for similar symmetric squaraines. The degenerate 2PA spectrum of 1 was obtained over a broad spectral range under femtosecond excitation, using standard open-aperture Z-scan and two-photon induced fluorescence methods, revealing maximum 2PA cross sections of ∼400 GM. Squaraine 1 exhibited efficient superluminescence emission in the polar solvent (dichloromethane) at room temperature under femtosecond pumping conditions. Quantum chemical analysis of the electronic structure of 1 was performed using the DFT/TD-DFT level of theory and found to be in good agreement with experimental data. The new squaraine derivative 1 displayed high fluorescence quantum yield, efficient NIR superluminescence, large 2PA cross sections, and high photostability with a photodecomposition quantum yield ∼4 × 10-6, suggesting its potential for applications in two-photon fluorescent bioimaging and lasing.

Folate receptor-targeted aggregation-enhanced near-IR emitting silica nanoprobe for one-photon in vivo and two-photon ex vivo fluorescence bioimaging.

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 µm deep in the HeLa tumor.

Two-photon fluorescence vascular bioimaging with new bioconjugate probes selective toward the vascular endothelial growth factor receptor 2.

We report the synthesis and characterization of two amine reactive fluorescent dyes with efficient two-photon absorption (2PA) properties and high fluorescence quantum yields. Bioconjugation of these dyes with the DC-101 antibody proved to be useful for selectively imaging the vascular endothelial growth factor receptor 2 (VEGFR-2) in cells expressing this receptor in vitro and in "whole" mounted excised tumors (ex vivo) by two-photon fluorescence microscopy (2PFM). The penetration depths reached within the tumors by 2PFM was over 800 µm. In addition, the concentration of dye required for incubation of these bioconjugates was in the picomolar domain, the probes possessed very good photostability, and the 2PFM setup did not require any additional means of increasing the collection efficiencies of fluorescent photons to achieve the relatively deep tissue imaging that was realized, due, in large part, to the favorable photophysical properties of the new probes.

Two-photon STED spectral determination for a new V-shaped organic fluorescent probe with efficient two-photon absorption.

Two-photon stimulated emission depletion (STED) cross sections were determined over a broad spectral range for a novel two-photon absorbing organic molecule, representing the first such report. The synthesis, comprehensive linear photophysical, two-photon absorption (2PA), and stimulated emission properties of a new fluorene-based compound, (E)-2-{3-[2-(7-(diphenylamino)-9,9-diethyl-9H-fluoren-2-yl)vinyl]-5-methyl-4-oxocyclohexa-2,5-dienylidene} malononitrile (1), are presented. Linear spectral parameters, including excitation anisotropy and fluorescence lifetimes, were obtained over a broad range of organic solvents at room temperature. The degenerate two-photon absorption (2PA) spectrum of 1 was determined with a combination of the direct open-aperture Z-scan and relative two-photon-induced fluorescence methods using 1 kHz femtosecond excitation. The maximum value of the 2PA cross section ~1700 GM was observed in the main, long wavelength, one-photon absorption band. One- and two-photon stimulated emission spectra of 1 were obtained over a broad spectral range using a femtosecond pump-probe technique, resulting in relatively high two-photon stimulated emission depletion cross sections (~1200 GM). A potential application of 1 in bioimaging was demonstrated through one- and two-photon fluorescence microscopy images of HCT 116 cells incubated with micelle-encapsulated dye.

Two-photon fluorescence lysosomal bioimaging with a micelle-encapsulated fluorescent probe.

We report two-photon fluorescence microscopy (2PFM) imaging and in vitro cell viability of a new, efficient, lysosome-selective system based on a two-photon absorbing (2PA) fluorescent probe (I) encapsulated in Pluronic® F-127 micelles. Preparation of dye I was accomplished via microwave-assisted synthesis, resulting in improved yields and reduced reaction times. Photophysical characterization revealed notable 2PA efficiency of this probe.

A superfluorescent fluorenyl probe with efficient two-photon absorption.

The linear photophysical, excited state absorption (ESA), superfluorescence, and two-photon absorption (2PA) properties of 4,4'-(1E,1'E)-2,2'-(7,7'(1E,1'E)2,2'(4,4'-sulfonylbis(4,1-phenylene))bis(ethane-2,1-diyl)bis(9,9-didecy-9H-fluorene7,2-diyl))bis(ethane-2,1-diyl)bis(N,N-diphenylaniline) (1) were investigated in organic and aqueous media with respect to its potential application in biological imaging. The analysis of linear photophysical properties revealed a rather complex nature of the main one-photon absorption band, strong solvatochromic effects in the steady-state fluorescence spectra, single-exponential fluorescence decay, and high fluorescence quantum yields in organic solvents (≈1.0). The ESA spectra of 1 suggested potential for light amplification in nonpolar media while efficient superfluorescence in cyclohexane was demonstrated. The degenerate 2PA spectra of 1 were obtained over a broad spectral range (640-900 nm), using a standard two-photon induced fluorescence method under 1 kHz femtosecond excitation. Two well defined 2PA bands with maximum 2PA cross sections up to 1700 GM in the higher energy, short wavelength band and ≈1200 GM in the lower energy, long wavelength band of 1 were shown. The potential use of 1 in bioimaging was demonstrated via one- and two-photon in vitro fluorescence imaging of HCT 116 cells.

Nature of Fast Relaxation Processes and Spectroscopy of a Membrane-Active Peptide Modified with Fluorescent Amino Acid Exhibiting Excited State Intramolecular Proton Transfer and Efficient Stimulated Emission.

A fluorescently labeled peptide that exhibited fast excited state intramolecular proton transfer (ESIPT) was synthesized, and the nature of its electronic properties was comprehensively investigated, including linear photophysical and photochemical characterization, specific relaxation processes in the excited state, and its stimulated emission ability. The steady-state absorption, fluorescence, and excitation anisotropy spectra, along with fluorescence lifetimes and emission quantum yields, were obtained in liquid media and analyzed based on density functional theory quantum-chemical calculations. The nature of ESIPT processes of the peptide's chromophore moiety was explored using a femtosecond transient absorption pump-probe technique, revealing relatively fast ESIPT velocity (∼10 ps) in protic MeOH at room temperature. Efficient superluminescence properties of the peptide were realized upon femtosecond excitation in the main long-wavelength absorption band with a corresponding threshold of the pump pulse energy of ∼1.5 µJ. Quantum-chemical analysis of the electronic structure of the peptide was performed using the density functional theory/time-dependent density functional theory level of theory, affording good agreement with experimental data.

High-fidelity hydrophilic probe for two-photon fluorescence lysosomal imaging.

The synthesis and characterization of a novel two-photon-absorbing fluorene derivative, LT1, selective for the lysosomes of HCT 116 cancer cells, is reported. Linear and nonlinear photophysical and photochemical properties of the probe were investigated to evaluate the potential of the probe for two-photon fluorescence microscopy (2PFM) lysosomal imaging. The cytotoxicity of the probe was investigated to evaluate the potential of using this probe for live two-photon fluorescence biological imaging applications. Colocalization studies of the probe with commercial Lysotracker Red in HCT 116 cells demonstrated the specific localization of the probe in the lysosomes with an extremely high colocalization coefficient (0.96). A figure of merit was introduced to allow comparison between probes. LT1 has a number of properties that far exceed those of commercial lysotracker probes, including higher two-photon absorption cross sections, good fluorescence quantum yield, and, importantly, high photostability, all resulting in a superior figure of merit. 2PFM was used to demonstrate lysosomal tracking with LT1.

Linear and nonlinear photophysics and bioimaging of an integrin-targeting water-soluble fluorenyl probe.

Linear photophysical characterization and two-photon absorption (2PA) properties of a new water-soluble fluorene derivative, 3-(9-(2-(2-methoxyethoxy)ethyl)-2,7-bis{3-[2-(polyethyleneglycol-550-monomethylether-1-yl)]-4-(benzo[d]thiazol-2-yl)styryl}-9H-fluoren-9-yl)propanoic acid (1), were investigated in several organic solvents and water at room temperature. A comprehensive analysis of the steady-state absorption, emission and excitation anisotropy spectra revealed electronic structures of 1, including mutual orientation of the transition dipoles, relatively weak solvatochromic effects, high fluorescence quantum yield (approximately 0.5-1.0), and strong aggregation in water. The 2PA spectra of 1 were obtained in the 600-900 nm spectral range by two-photon induced fluorescence (2PF) and open aperture Z-scan methods using femtosecond laser sources. No discrete 2PA bands were apparent and values of the corresponding 2PA cross sections monotonically increased in the short wavelength range up to 3000 GM in organic solvents and approximately 6000 GM in aqueous solution, reflecting relatively high two-photon absorptivity. The 2PA efficiency of in water increased 2-3 times relative to aprotic solvents and can be explained by cooperative electronic effects of molecular aggregates of 1 produced in aqueous media. The carboxylic acid fluorenyl probe 1 was conjugated with the cyclic peptide RGDfK. Two-photon fluorescence microscopy (2PFM) imaging of U87MG cells (and MCF-7 as control), incubated with fluorene-RGD peptide conjugate 2, demonstrated high alpha(v)beta(3) integrin selectivity, making this probe particularly attractive for integrin imaging.

Efficient two-photon absorbing acceptor-pi-acceptor polymethine dyes.

We present an experimental and theoretical investigation of the linear and nonlinear optical properties of a series of acceptor-pi-acceptor symmetrical anionic polymethine dyes with diethylamino-coumarin-dioxaborine terminal groups and different conjugation lengths. Two-photon absorption (2PA) cross sections (delta(2PA)) are enhanced with an increase of pi-conjugation length in the investigated series of dyes. 2PA spectra for all dyes consist of two well-separated bands. The first band, located within the telecommunications window, occurs upon two-photon excitation into the vibrational levels of the main S(0) --> S(1) transition, reaching a large delta(2PA) = 2200 GM (1 GM = 1 x 10(-50) cm(4) s/photon) at 1600 nm for the longest conjugated dye. The position of the second, and strongest, 2PA band for all anionic molecules corresponds to the second-excited final state, which is confirmed by quantum-chemical calculations and excitation anisotropy measurements. Large delta(2PA) values up to 17,000 GM at 1100 nm are explained by the combination of the large ground- and excited-state transition dipole moments. The three shortest dyes show good photochemical stability and surprisingly large fluorescence quantum yields of approximately 0.90, approximately 0.66, and approximately 0.18 at the red to near-IR region of approximately 640, approximately 730, and approximately 840 nm, respectively. The excited-state absorption spectra for all samples are also studied and exhibit intense bands throughout the visible wavelength region with peak cross section close to 5 x 10(-16) cm(2) with a corresponding red shift with increasing conjugation lengths.