Two-photon Excited Emission Research Articles

Isomeric Effect of Mercaptobenzoic Acids on the Synthesis, Stability, and Optical Properties of Au25(MBA)18 Nanoclusters.

We report a simple size focusing, two-step “bottom-up” protocol to prepare water-soluble Au25(MBA)18 nanoclusters, using the three isomers of mercaptobenzoic acids (p/m/o-MBA) as capping ligands and Me3NBH3 as a mild reducing agent. The relative stability of the gas-phase multiply deprotonated Au25(MBA)18 ions was investigated by collision-induced dissociation. This permitted us to evaluate the possible isomeric effect on the Au–S interfacial bond stress. We also investigated their optical properties. The absorption spectra of Au25(MBA)18 isomers were very similar and showed bands at 690, 470, and 430 nm. For all Au25(MBA)18 isomeric clusters, no measurable one-photon excited fluorescence under UV–vis light was found, in neither solid- nor solution-state. The two-photon excited emission spectra and first hyperpolarizabilities of the clusters were also determined. The results are discussed in terms of the possible isomeric effect on excitations within the metal core and the possibility of charge transfer excitations from the ligands to the metal nanocluster.

Two-photon excitation and direct emission from S2 state of U.S. Food and Drug Administration approved near-infrared dye: Application of anti-Kasha's rule for two-photon fluorescence imaging.

In recent years, two-photon fluorescence microscopy has gained significant interest in bioimaging. It allows the visualization of deeply buried inhomogeneities in tissues. The near-infrared (NIR) dyes are also used for deep tissue imaging. Indocyanine green (ICG) is the only U.S. Food and Drug Administration (FDA) approved exogenous contrast agent in the NIR region for clinical applications. However, despite its potential candidature, it had never been used as a two-photon contrast agent for biomedical imaging applications. This letter provides an insight into the scope and application of the two-photon excitation property of ICG to the second excited singlet (S2 ) state in aqueous solution. Furthermore, in this work, we demonstrate the two-photon cellular imaging application of ICG using direct fluorescence emission from S2 state for the first time. Our results show that two-photon excitation to S2 state of ICG could be achieved with approximately 790 nm wavelength of femtosecond laser, which lies in well-known "tissue-optical window." This property would enable light to penetrate much deeper in the turbid medium such as biological tissues. Thus, ICG could be used as the first FDA approved NIR exogenous contrast agent for two-photon imaging. These findings can make remarkable influence on preclinical and clinical cell imaging.

Two-photon isomerization triggers two-photon-excited fluorescence of an azobenzene derivative

Photoisomerization provides a general means for photo-controlling molecular structure and function, and two-photon induced isomerization has some advantages over one-photon processes. Here, we report the two-photon absorption (TPA) induced optical properties and characteristics of an azobenzene derivative, namely N-(3,4,5-octanoxyphenyl)-N′-4-[(4-hydroxyphenyl)azophenyl]1,3,4-oxadiazole (AOB-t8). With the activation of red light, the photoproduct of cis-AOB-t8 isomers has been unambiguously identified, and the two-photon induced isomerization process of AOB-t8 in THF is characterized with the relevant rate constants of forward and backward reactions. The TPA coefficients and cross-sections of AOB-t8 at different wavelengths, and nonlinear optical refractive index are determined from femtosecond nonlinear optical measurements. Particularly, the results indicate that the two-photon excited emission can be effectively triggered by the two-photon-induced isomerization from trans- to cis-AOB-t8, demonstrating an on-switch from non-fluorescence to visible blue emission. Consistent with the observations from one-photon excitation, we suggest that the enhanced two-photon excited fluorescence of AOB-t8 solution originates from the combined contribution of cis-AOB-t8 monomer and its aggregates, i.e., the conformation transition from trans- to cis-AOB-t8 opens the emission channel and the self-assembly aggregation of cis-AOB-t8 isomers further enhances the fluorescence upon red light excitation.

Preparation of a Nile Red-Pd-based fluorescent CO probe and its imaging applications in vitro and in vivo.

Carbon monoxide (CO) is a key gaseous signaling molecule in living cells and organisms. This protocol illustrates the synthesis of a highly sensitive Nile Red (NR)-Pd-based fluorescent probe, NR-PdA, and its applications for detecting endogenous CO in tissue culture cells, ex vivo organs, and zebrafish embryos. In the NR-PdA synthesis process, 3-diethylamine phenol reacts with sodium nitrite in the acidic condition to afford 5-(diethylamino)-2-nitrosophenol hydrochloride (compound 1), which is further treated with 1-naphthalenol at a high temperature to provide the NR dye via a cyclization reaction. Finally, NR is reacted with palladium acetate to obtain the desired Pd-based fluorescent probe NR-PdA. NR-PdA possesses excellent two-photon excitation and near-IR emission properties, high stability, low background fluorescence, and a low detection limit. In addition to the chemical synthesis procedures, we provide step-by-step procedures for imaging endogenous CO in RAW 264.7 cells, mouse organs ex vivo, and live zebrafish embryos. The synthesis process for the probe requires ∼4 d, and the biological imaging experiments take ∼14 d.

Cancer Cell Membrane-Biomimetic Nanoprobes with Two-Photon Excitation and Near-Infrared Emission for Intravital Tumor Fluorescence Imaging.

Biomimetic fluorescent nanoprobes capable of emitting near-infrared (NIR) fluorescence (λmax ≈ 720 nm) upon excitation of 800 nm light were developed. The key conjugated polymer enabled two-photon absorption and Förster resonance energy transfer (FRET) processes within the nanoprobes, which imparted the nanoprobes with ideal NIR-incoming-NIR-outgoing fluorescence features. The cancer cell membrane (CM) coating endowed these nanoprobes with perfect biocompatibility and highly specific targeting ability to homologous tumors. It was believed that CM encapsulation provided an additional protecting layer for the photoactive components residing in the core of nanoprobes for retaining their intrinsic fluorescing ability in the physiological milieu. The long-term structural integrity, excellent photostability (fluorescence decrease <10% upon 30 min illumination of 800 nm pulse laser), high NIR fluorescence quantum yield (∼20%), and long in vivo circulation time of the target nanoprobes were also confirmed. The ability of these feature-packed nanoprobes for circumventing the challenges of absorption and light scattering caused by cellular structures and tissues was definitely confirmed via in vivo and in vitro experiments. The superior performances of these nanoprobes in terms of fluorescence signaling as well as targeting specificity were verified in intravital fluorescence imaging on tumor-bearing model mice. Specifically, these nanoprobes unequivocally enabled high-resolution visualization of the fine heterogeneous architectures of intravital tumor tissue, which proclaims the great potential of this type of probe for high-contrast fluorescence detection of thick biological samples in practical applications.

Atomically precise clusters of gold and silver: A new class of nonlinear optical nanomaterials

Functional ligand-protected metal cluster nanomaterials with increased two-photon absorption and two-photon excited emission might result in new technologies within the fields of bio-imaging applications. During this article, I review the two-photon absorption/emission properties of atomically precise ligand-protected silver and gold nanoclusters, coined “ligand-core” NLO-phores. This includes quantum-chemical methodologies using time-dependent density theory. I will analyze physical phenomena and trends resulting in giant two-photon absorption/emission responses of a number of series of model nanoclusters. I will then focus my review on the effects of the relaxation pathways within the linear and nonlinear optical regime, similarly as innovative ways aiming at enhancing their two-photon emission responses.

Tuning Two-Photon Absorption Cross Section in Metal Organic Frameworks

The development of alternative nonlinear optical metamaterials has attracted much attention recently due to technological demands. Upconversion emission via a simultaneous two-photon absorption process is a nonlinear process that is widely studied in synthetically challenging organic compounds. Hereby, we report 9 metal organic frameworks constructed with various combinations of the following ligands: trans,trans-9,10-bis(4-pyridylethenyl) anthracene, trans,trans-9,10-bis(4-pyridylethynyl) anthracene, 1,4-bis[2-(4′-pyridyl)ethenyl]benzene, 4,4′-stilbene dicarboxylate, 4,4′-biphenyl dicarboxylate, 4,4′-benzene dicarboxylate, and benzene-1,3,5-tricarboxylate. Altering the auxiliary carboxylate ligands not only changes the structure but also varies the two-photon excited fluorescence. The two-photon excited emission is enhanced when longer spacer ligands are used and when they are packed in more expanded structures in hms topology. Unusually, the emission becomes stronger when a pair of pyridyl type ligands ...

Optical Properties and Structural Relationships of the Silver Nanoclusters Ag32(SG)19 and Ag15(SG)11

The recent discovery of stable Ag nanoclusters presents new opportunities to understand the detailed electronic and optical properties of the metal core and the ligands using ultrafast spectroscopy. This paper focuses on Ag32 and Ag15 (with thiolate ligands), which are stable in solution. The steady state absorption spectra of Ag nanoclusters show interesting quantum size effects, expected for this size regime. Using a simple structural model for Ag32, TDDFT calculations show absorption at 480 nm and 680 nm that are in reasonable correspondence with experiments. Ag32(SG)19 and Ag15(SG)11 have quantum yields up to 2 orders of magnitude higher than Au nanoclusters of similar sizes, with an emission maximum at 650 nm, identified as the metal–ligand state. The emission from both Ag nanoclusters has a common lifetime of about 130 ps and a common energy transfer rate of KEET ≥ 9.7 × 109 s–1. A “dark state” competing with the emission process was also observed and was found to be directly related to the difference in quantum yield (QY) for the two Ag clusters. Two-photon excited emission was observed for Ag15(SG)11, with a cross-section of 34 GM under 800 nm excitation. Femtosecond transient absorption measurements for Ag32 recorded a possible metal core state at 530 nm, a metal–ligand state at 651 nm, and ground state bleaches at 485 and 600 nm. The ground state bleach signals in the transient spectrum for Ag32 are 100 nm blue-shifted in comparison to Au25. The transient spectrum for Ag15 shows a weak ground state bleach at ∼480 nm and a broad excited state centered at 610 nm. TDDFT calculations indicate that the electronic and optical properties of Ag nanoclusters can be divided into core states and metal–ligand states, and photoexcitation generally involves a ligand to metal core transition. Subsequent relaxation leaves the electron in a core state, but the hole can be either ligand or core-localized. This leads to emission/relaxation that is consistent with the observed photophysics.

Ligand-core NLO-phores: a combined experimental and theoretical approach to the two-photon absorption and two-photon excited emission properties of small-ligated silver nanoclusters.

We report a combined experimental and theoretical study of the two-photon absorption and excited emission properties of monodisperse ligand stabilized Ag11, Ag15 and Ag31 nanoclusters in aqueous solutions. The nanoclusters were synthesized using a cyclic reduction under oxidative conditions and separated by vertical gel electrophoresis. The two-photon absorption cross-sections of these protected noble metal nanoclusters measured within the biologically attractive 750-900 nm window are several orders of magnitude larger than that reported for commercially available standard organic dyes. The two-photon excited fluorescence spectra are also presented for excitation wavelengths within the same excitation spectral window. They exhibit size-tunability. Because the fundamental photophysical mechanisms underlying these multiphoton processes in ligand protected clusters with only a few metal atoms are not fully understood yet, a theoretical model is proposed to identify the key driving elements. Elements that regulate the dipole moments and the nonlinear optical properties are the nanocluster size, its structure and the charge distribution on both the metal core and the bound ligands. We coined this new class of NLO materials as "Ligand-Core" NLO-phores.

An easily accessible aggregation-induced emission probe for lipid droplet-specific imaging and movement tracking

Lipid droplets (LDs) as dynamic organelles are associated with many metabolic processes. Ideal fluorescent probes for LD-specific imaging require excellent specificity, superior brightness, fast cell permeability, and easy preparation. However, conventional fluorophores for LD imaging suffer from drawbacks of aggregation-caused quenching (ACQ), poor photostability, and difficulty of preparation. To tackle these challenges, herein, we develop an easily accessible aggregation-induced emission (AIE) fluorescent probe for LD-specific imaging and dynamic movement tracking. This AIE probe has significant advantages in terms of fast cell permeability, low cytotoxicity, strong photostability, and high two-photon absorption cross-sections in the near infra-red (NIR) range. It is thus expected to have broad applications in the study of LDs' biological functions.

An N-nitrosation reactivity-based two-photon fluorescent probe for the specific in situ detection of nitric oxide.

In situ fluorescence imaging of nitric oxide (NO) is a powerful tool for studying the critical roles of NO in biological events. However, the selective imaging of NO is still a challenge because most currently available fluorescent probes rely on the o-phenylenediamine (OPD) recognition site, which reacts with both NO and some abundant reactive carbonyl species (RCS) (such as dehydroascorbic acid and methylglyoxal) and some reactive oxygen/nitrogen species (ROS/RNS). To address this problem, a new fluorescent probe, NCNO, based on the N-nitrosation of aromatic secondary amine was designed to bypass the RCS, ROS, and RNS interference. As was expected, the probe NCNO could recognize NO with pronounced selectivity and sensitivity among ROS, RNS, and RCS. The probe was validated by detecting NO in live cells and deep tissues owing to its two-photon excitation and red-light emission. It was, hence, applied to monitor NO in ischemia reperfusion injury (IRI) in mice kidneys by two-photon microscopy for the first time, and the results vividly revealed the profile of NO generation in situ during the renal IRI process.

Two-photon absorption in penicillamine capped CdS tetrapods

Synthesized penicillamine stabilized CdS tetrapods showed two-photon absorption in a wavelength range between 600 and 850 nm, and strong two-photon excited emission upon near infrared excitation. These water soluble colloidal, semiconducting nanoparticles show potential for applications in nonlinear bioimaging.

Solvent effect on the femtosecond laser induced two-photon emission from Rhodamine 6G

Abstract Two-photon fluorescence microscopy attains widespread importance because of its applications in biological imaging. This technology provides a non-invasive study of biological specimens with better resolution in three dimensions with less photo damage. We report on two-photon excited emission from a widely used laser dye Rhodamine 6G in five different polar solvents using a mode-locked Ti: Sapphire pulsed laser. Solvent plays a major role in the photo physical properties of the lasing dye. There is a shift in the peak fluorescence wavelength as the concentration is varied. A solvent based shift in peak fluorescence wavelength is also observed. Maximum peak intensity is observed for the sample in the solvent ethanol. It was found that lifetime is varied depending on the nature of the solvent used.

Self-Assembly of Electron Donor-Acceptor-Based Carbazole Derivatives: Novel Fluorescent Organic Nanoprobes for Both One- and Two-Photon Cellular Imaging.

In this study, we report fluorescent organic nanoprobes with intense blue, green, and orange-red emissions prepared by self-assembling three carbazole derivatives into nanorods/nanoparticles. The three compounds consist of two or four electron-donating carbazole groups linked to a central dicyanobenzene electron acceptor. Steric hindrance from the carbazole groups leads to noncoplanar 3D molecular structures favorable to fluorescence in the solid state, while the donor-acceptor structures endow the molecules with good two-photon excited emission properties. The fluorescent organic nanoprobes exhibit good water dispersibility, low cytotoxicity, superior resistance against photodegradation and photobleaching. Both one- and two-photon fluorescent imaging were shown in the A549 cell line. Two-photon fluorescence imaging with the fluorescent probes was demonstrated to be more effective in visualizing and distinguishing cellular details compared to conventional one-photon fluorescence imaging.

Ethylene glycol modified 2-(2′-aminophenyl)benzothiazoles at the amino site: the excited-state N-H proton transfer reactions in aqueous solution, micelles and potential application in live-cell imaging

Triethylene glycol monomethyl ether and poly(ethylene glycol) monomethyl ether modified 2-(2′-aminophenyl)benzothiazoles, namely ABT-P3EG, ABT-P7EG and ABT-P12EG varied by different chain length of poly(ethylene glycol) at the amino site, were synthesized to probe their photophysical and bio-imaging properties. In polar, aprotic solvents such as CH2Cl2 ultrafast excited-state intramolecular proton transfer (ESIPT) takes place, resulting in a large Stokes shifted tautomer emission in the green-yellow (550 nm) region. In neutral water, ABT-P12EG forms micelles with diameters of 15 ± 3 nm under a critical micelle concentration (CMC) of ~80 μM, in which the tautomer emission is greatly enhanced free from water perturbation. Cytotoxicity experiments showed that all ABT-PnEGs have negligible cytotoxicity against HeLa cells even at doses as high as 1 mM. Live-cell imaging experiments were also performed, the results indicate that all ABT-PnEGs are able to enter HeLa cells. While the two-photon excitation emission of ABT-P3EG in cells cytoplasm shows concentration independence and is dominated by the anion blue fluorescence, ABT-P7EG and ABT-P12EG exhibit prominent green tautomer emission at > CMC and in part penetrate to the nuclei, adding an additional advantage for the cell imaging.

Optical nonlinearities of colloidal InP@ZnS core-shell quantum dots probed by Z-scan and two-photon excited emission

Spectrally resolved nonlinear optical properties of colloidal InP@ZnS core-shell quantum dots of various sizes were investigated with the Z-scan technique and two-photon fluorescence excitation method using a femtosecond laser system tunable in the range from 750 nm to 1600 nm. In principle, both techniques should provide comparable results and can be interchangeably used for determination of the nonlinear optical absorption parameters, finding maximal values of the cross sections and optimizing them. We have observed slight differences between the two-photon absorption cross sections measured by the two techniques and attributed them to the presence of non-radiative paths of absorption or relaxation. The most significant value of two-photon absorption cross section σ2 for 4.3 nm size InP@ZnS quantum dot was equal to 2200 GM, while the two-photon excitation action cross section σ2Φ was found to be 682 GM at 880 nm. The properties of these cadmium-free colloidal quantum dots can be potentially useful for nonlinear bioimaging.

Mechanism of two-photon excited hemoglobin fluorescence emission.

Hemoglobin, one of the most important proteins in the human body, is composed of “heme” groups (iron-containing rings) and “globins” (proteins). We investigate the two-photon excited fluorescence of hemoglobin and its subunit components (heme and globin). We measure the hemoglobin fluorescence lifetime by using a streak camera of ps resolution and confirm that its lifetime is in femtosecond scale. In the study of the fluorescence properties of heme and globin, the experimental results reveal that heme is the sole fluorophore of hemoglobin. Hemoglobin fluorescence can be effectively excited only via two-photon process, because heme has a centrosymmetric molecular structure and two-photon allowed transition is forbidden for single-photon process and vice versa due to the Laporte parity selection rule.

Conjugated Polymer-Based Hybrid Nanoparticles with Two-Photon Excitation and Near-Infrared Emission Features for Fluorescence Bioimaging within the Biological Window.

Hybrid fluorescent nanoparticles (NPs) capable of fluorescing near-infrared (NIR) light (centered ∼730 nm) upon excitation of 800 nm laser light were constructed. A new type of conjugated polymer with two-photon excited fluorescence (TPEF) feature, P-F8-DPSB, was used as the NIR-light harvesting component and the energy donor while a NIR fluorescent dye, DPA-PR-PDI, was used as the energy acceptor and the NIR-light emitting component for the construction of the fluorescent NPs. The hybrid NPs possess δ value up to 2.3 × 10(6) GM per particle upon excitation of 800 nm pulse laser. The excellent two-photon absorption (TPA) property of the conjugated polymer component, together with its high fluorescence quantum yield (ϕ) up to 45% and the efficient energy transfer from the conjugated polymer to NIR-emitting fluorophore with efficiency up to 90%, imparted the hybrid NPs with TPEF-based NIR-input-NIR-output fluorescence imaging ability with penetration depth up to 1200 μm. The practicability of the hybrid NPs for fluorescence imaging in Hela cells was validated.

Two-Photon Spectroscopy as a New Sensitive Method for Determining the DNA Binding Mode of Fluorescent Nuclear Dyes.

A new optical strategy to determine the binding modes (intercalation vs groove binding) of small fluorescent organic molecules with calf thymus DNA was developed using two-photon absorption (TPA) spectroscopy. Two-photon excited emission was utilized to investigate a series of fluorescent nuclear dyes. The results show that TPA cross-sections are able to differentiate the fine details between the DNA binding modes. Groove binding molecules exhibit an enhanced TPA cross-section due to the DNA electric field induced enhancement of the transition dipole moment, while intercalative binding molecules exhibit a decrease in the TPA cross-section. Remarkably, the TPA cross-section of 4,6-bis(4-(4-methylpiperazin-1-yl)phenyl) pyrimidine is significantly enhanced (13.6-fold) upon binding with DNA. The sensitivity of our TPA methodology is compared to circular dichroism spectroscopy. TPA demonstrates superior sensitivity by more than an order of magnitude at low DNA concentrations. This methodology can be utilized to probe DNA interactions with other external molecules such as proteins, enzymes, and drugs.

Highly efficient, conjugated-polymer-based nano-photosensitizers for selectively targeted two-photon photodynamic therapy and imaging of cancer cells.

Two-photon photodynamic therapy (2P-PDT) is a promising noninvasive treatment of cancers and other diseases with three-dimensional selectivity and deep penetration. However, clinical applications of 2P-PDT are limited by small two-photon absorption (TPA) cross sections of traditional photosensitizers. The development of folate receptor targeted nano-photosensitizers based on conjugated polymers is described. In these nano-photosensitizers, poly{9,9-bis[6''-(bromohexyl)fluorene-2,7-ylenevinylene]-co-alt-1,4-(2,5-dicyanophenylene)}, which is a conjugated polymer with a large TPA cross section, acts as a two-photon light-harvesting material to significantly enhance the two-photon properties of the doped photosensitizer tetraphenylporphyrin (TPP) through energy transfer. These nanoparticles displayed up to 1020-fold enhancement in two-photon excitation emission and about 870-fold enhancement in the two-photon-induced singlet oxygen generation capability of TPP. Surface-functionalized folic acid groups make these nanoparticles highly selective in targeting and killing KB cancer cells over NIH/3T3 normal cells. The 2P-PDT activity of these nanoparticles was significantly improved, potentially up to about 1000 times, as implied by the enhancement factors of two-photon excitation emission and singlet oxygen generation. These nanoparticles could act as novel two-photon nano-photosensitizers with combined advantages of low dark cytotoxicity, targeted 2P-PDT with high selectivity, and simultaneous two-photon fluorescence imaging capability; these are all required for ideal two-photon photosensitizers.