Strong Two-photon Excited Fluorescence Research Articles

Two-Photon Near-Infrared AIE Luminogens as Multifunctional Gene Carriers for Cancer Theranostics.

Construction of multifunctional nonviral gene vectors to execute defined tasks holds great potential for the precise and effective treatment of gene-associated diseases. Herein, we have developed four large π-conjugation triphenylamine derivatives bearing two polar [12]aneN3 heads and a lipophilic tail for applications in gene delivery, one/two-photon-triggered near-infrared (NIR) fluorescence bioimaging, and combined photodynamic therapy (PDT) and gene therapy of cancer. These compounds possess typical NIR aggregation-induced emission characteristics, mega Stokes shifts, strong two-photon excitation fluorescence, and excellent DNA condensation abilities. Among them, vector 4 with a tail of n-hexadecane realized a transfection efficiency as high as 6.7 times that of the commercial transfection agent Lipofectamine 2000 in HEK293T cell lines. Using vector 4 as an example, transfection process tracking and ex vivo/in vivo tumoral imaging and retention with high resolution, high brightness, deep tissue penetration, and good biosafety were demonstrated. In addition, efficient singlet oxygen (1O2) generation by the DNA complex formed by vector 4 (4/DNA) resulted in effective PDT. Combined with anticancer gene therapy, collaborative cancer treatment with a dramatically enhanced cancer cell-killing effect was achieved. The development of this "three birds, one stone" approach suggests a new and promising strategy for better cancer treatment and real-time tracking of gene delivery.

Naphthalimide-based probe with strong two-photon excited fluorescence and high specificity to cell membranes

By introducing a long alkyl chain and a quaternary ammonium on the two sides of naphthalimide fluorophore, we have developed an amphiphilic probe NA with a high specificity toward cell membranes and strong two-photon excited fluorescence. Due to these advantageous features, NA demonstrates wash-free staining and high quality two-photon imaging of live cells.

Synthesis and two-photon properties of phenothiazine-based dendrimers with tetraphenylmethane cores

We have synthesized two new two-photon active dendritic molecules with tetraphenylmethane core with four phenothiazine-based arms. They show good solubility in organic solvents, including dichloromethane, chloroform, toluene and tetrahydrofuran. It is found that the two dendrimers display strong two-photon absorption and two-photon excited up-conversion fluorescence at 800 nm. • Two new phenothiazine-based dendrimers with tetraphenylmethane cores have been synthesized. • The dendritic molecules display two-photon absorption activity. • The two-photon absorption cross section increased with the increasing of the conjugation degree.

Cooperation of ESIPT and ICT Processes in the Designed 2-(2'-Hydroxyphenyl)benzothiazole Derivative: A Near-Infrared Two-Photon Fluorescent Probe with a Large Stokes Shift for the Detection of Cysteine and Its Application in Biological Environments.

A rationally designed near-infrared two-photon fluorescent probe (SDP-A) for selectively detecting cysteine (Cys) has been developed based on a newly designed conjugation-enhanced 2-(2'-hydroxyphenyl)benzothiazole derivative as the fluorophore, an acrylate moiety as the Cys reaction site, and an N-methylpyridinium scaffold as both the unit of organelle targeting and improving water solubility. The probe SDP-A alone essentially emitted no fluorescence, whereas it achieved a superb near-infrared fluorescence emission (713 nm) enhancement within 15 min with a significant Stokes shift (302 nm) in the presence of Cys. The photoluminescence mechanism of the probe SDP-A toward Cys was modulated by excited-state intramolecular proton transfer (ESIPT) and intramolecular charge transfer (ICT) processes. It exhibited high selectivity and sensitivity (LOD = 102 nM) for monitoring Cys over other analytes such as Hcy/GSH/H2S owing to a specific conjugate addition-cyclization reaction between Cys and the acrylate moiety. More importantly, the released fluorophore SDP exhibits elevated quantum yields (1.52-18.17%) in different solvents and strong two-photon excited fluorescence with a sizeable two-photon action cross-section (Φ) of 213.5 GM at 820 nm in acetonitrile-PBS medium, which is highly desirable for two-photon fluorescence imaging of the living samples. Therefore, SDP-A was successfully applied to the imaging of Cys in live cells, zebrafish, mouse brain, and abdominal cavity down to a depth of more than 200 μm using a one/two-photon fluorescence microscope.

Two-photon excited luminescence and second-harmonic generation in quinacridone microstructures

Nonlinear optical microscopy was employed to investigate the properties of microstructures of quinacridone, a hydrogen-bonded pigment, derivative of quinacridine. Recently, quinacridone hierarchical microstructures (μ-QNC) having different nature-inspired morphologies were reported, e.g. in the shape of hedgehogs formed by self-assembled nano-needles. We find that such microcrystals have structure-dependent linear optical properties and exhibit strong two-photon excited fluorescence when irradiated with a near-infrared femtosecond laser. μ-QNCs exhibit different nonlinear optical properties for different crystal structures, including the emergence of symmetry-forbidden second-harmonic generation.

Targetable, two-photon fluorescent probes for local nitric oxide capture in the plasma membranes of live cells and brain tissues.

Although the plasma membrane is a major site for nitric oxide (NO) generation and action, few targetable probes that specifically sense and image NO in the plasma membrane have been reported. In this study, a membrane targetable, two-photon nitric oxide probe, Mem-NO, was developed and evaluated for bio-imaging of both exogenous and endogenous NO. By installing a quaternary ammonium compound as the hydrophilic head and a long alkyl chain as the hydrophobic tail on 4-amino-1,8-naphthalimide, we designed Mem-NO into a bi-polar structure. Due to the interaction with the phospholipid bilayer of plasma membrane, Mem-NO could specifically and stably localize in the plasma membrane. Mem-NO is almost nonfluorescent, but it displayed substantial fluorescence enhancement (16-fold) upon NO capture with sensitive (74 nM limit of detection) and fast response (within 1 min). Moreover, Mem-NO displayed strong two-photon excitation fluorescence activity (δ = 177 GM at 810 nm) and low cytotoxicity. It was found that Mem-NO is capable of two-photon imaging of endogenous NO in live neurons and human umbilical vein endothelial cells (HUVECs) and exogenous NO in mouse brain tissues. Therefore, Mem-NO qualifies as an essential and unique analytical tool for monitoring NO for future physiological, pathological, and pharmacological studies.

Two-photon absorption and mechanofluorochromic properties of 1,4-diketo-2,5-dibutyl-3,6-bis(4-(carbazol-N-yl)phenyl)pyrrolo[3,4-c]pyrrole

A new diketopyrrolopyrrole-based fluorescent dye 1,4-diketo-2,5-dibutyl-3,6-bis(4- (carbazol-N-yl)phenyl)pyrrolo[3,4-c]pyrrole (DPPCZ) was synthesized and characterized. DPPCZ with donor–π–acceptor–π–donor motif exhibited large and solvent-dependent two-photon absorption cross sections (δ) and emitted strong two-photon excitation fluorescence. The measured maximal δ values in CHCl3, THF, DMF, and toluene are 750, 610, 600, and 460g, respectively. While DPPCZ water-dispersions exhibit aggregation-quenched emission to some extent, the pristine powder, the obtained single crystal, and emulsion could still emit the relatively strong fluorescence with the efficiency of 21%, 9.5%, and 24%, respectively. It was found that both yellow and red DPPCZ crystals with weak intermolecular interactions could be ground into an amorphous orange state, and then recovered to the yellow state by thermal annealing and solvent-fuming, affording a new DPP-based mechanofluoro-chromic (MFC) material. Powder X-ray diffraction and differential scanning calorimetry indicated that the phase transition between crystalline and amorphous states upon external stimuli was responsible for the MFC behavior.

Preparation and Photophysical Properties of All-trans Acceptor–π-Donor (Acceptor) Compounds Possessing Obvious Solvatochromic Effects

A series of all-trans acceptor–π-donor (acceptor) compounds (BAQ, SFQ, BLQ, and XJQ) were conveniently synthesised and characterised by infrared, nuclear magnetic resonance, mass spectrometry, and elemental analysis. Their photophysical properties, including linear absorption, one-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence, were systematically investigated. All the compounds show obvious solvatochromic effects, such as significant bathochromic shifts of the emission spectra and larger Stokes shifts in more polar solvents. Under excitation from a femtosecond Ti : sapphire laser with a pulse width of 140 fs, they all exhibit strong two-photon excited fluorescence, and the two-photon absorption cross-sections in THF are 851 (BAQ), 216 (SFQ), 561 (BLQ), and 447 (XJQ) GM respectively. A combination of density functional theory (DFT) and time-dependent density functional theory (TDDFT) approaches was used to investigate the relationships between the structures and the photophysical properties of these compounds. The results show that they may have a potential application as polarity-sensitive two-photon fluorescent probes.

Highly Selective Two-Photon Fluorescent Probe for Ratiometric Sensing and Imaging Cysteine in Mitochondria

A novel ratiometric mitochondrial cysteine (Cys)-selective two-photon fluorescence probe has been developed on the basis of a merocyanine as the fluorophore and an acrylate moiety as the biothiol reaction site. The biocompatible and photostable acrylate-functionalized merocyanine probe shows not only a mitochondria-targeting property but also highly selective detection and monitoring of Cys over other biothiols such as homocysteine (Hcy) and glutathione (GSH) and hydrogen sulfide (H2S) in live cells. In addition, this probe exhibits ratiometric fluorescence emission characteristics (F518/F452), which are linearly proportional to Cys concentrations in the range of 0.5-40 μM. More importantly, the probe and its released fluorophore, merocyanine, exhibit strong two-photon excited fluorescence (TPEF) with two-photon action cross-section (Φσmax) of 65.2 GM at 740 nm and 72.6 GM at 760 nm in aqueous medium, respectively, which is highly desirable for high contrast and brightness ratiometric two-photon fluorescence imaging of the living samples. The probe has been successfully applied to ratiometrically image and detect mitochondrial Cys in live cells and intact tissues down to a depth of 150 μm by two-photon fluorescence microscopy. Thus, this ratiometric two-photon fluorescent probe is practically useful for an investigation of Cys in living biological systems.

Study of the one-photon and two-photon properties of two water-soluble terpyridines and their zinc complexes.

Two water soluble 2,2':6',2''-terpyridine derivatives (TPYOH and O3TPY) with a di(hydroxyethyl)amino or a long (2-(2-methoxyethoxy)ethoxy)benzyl group, respectively, were designed and synthesized. The two terpyridine ligands and their Zn(ii) complexes display strong one-photon fluorescence. An unusually large solvatochromism effect in ethanol and water was found, which was ascribed to their interactions with the solvent through hydrogen bonds. All the four compounds show evident two-photon absorption ability when evaluated by Z-scan technology. The free ligands could give distinct two-photon excited fluorescence, and the emission of O3TPY responded linearly with Zn(2+) under the excitation of a 780 nm femtosecond laser. Only the complex ZnO3TPY, which possesses a large π conjugation system and suitable election-donating groups, displayed emission maxima at 595 nm when excited by a 680-880 nm laser.

Theranostic iridium(III) complexes as one- and two-photon phosphorescent trackers to monitor autophagic lysosomes.

During autophagy, the intracellular components are captured in autophagosomes and delivered to lysosomes for degradation and recycling. Changes in lysosomal trafficking and contents are key events in the regulation of autophagy, which has been implicated in many physiological and pathological processes. In this work, two iridium(III) complexes (LysoIr1 and LysoIr2) are developed as theranostic agents to monitor autophagic lysosomes. These complexes display lysosome-activated phosphorescence and can specifically label lysosomes with high photostability. Simultaneously, they can induce autophagy potently without initiating an apoptosis response. We demonstrate that LysoIr2 can effectively implement two functions, namely autophagy induction and lysosomal tracking, in the visualization of autophagosomal-lysosomal fusion. More importantly, they display strong two-photon excited fluorescence (TPEF), which is favorable for live cell imaging and in vivo applications.

New conjugated organic dyes with various electron donors: One- and two-photon excited fluorescence, and bioimaging

Six new fluorescent donor-π bridge-acceptor (D-π-A) structural molecules with various electron donors were synthesized and fully characterized. Influence of electron-donating strength on optical properties was investigated by the systematic alteration of the electron donors. The optical properties of the dyes were successfully tuned by choosing different electron donors. All the dyes in solution show strong one-photon excited fluorescence (OPEF) and high quantum yield. With increasing electron-donating strength of donors, dye 5 with diphenylamine and dye 6 with (4,4′-diethoxyphenyl)amino substituents, exhibit strong two-photon excited fluorescence (TPEF), and the large two-photon absorption (TPA) cross-section values are 1378 GM for 5 and 1654 GM for 6. The structure–property relationships were detailed through quantum chemical calculations and X-ray crystallography. The results of living cell imaging experiments show the values of them in one- or two-photon fluorescence microscopy bioimaging applications.

Two-photon absorption and fluorescence fluoride-sensing properties of N-octyl-3,6-bis[4-(4-(diphenylamino)phenyl)phenyl]-1,4-diketo-pyrrolo[3,4-c]pyrrole

A fully aromatic D–π–A–π–D-type molecule 3,6-bis[4-(4-(diphenylamino)-phenyl)phenyl]-N-octyl-1,4-diketo-pyrrolo[3,4-c]pyrrole (TDP) was synthesized, and the one- and two-photon absorption and emission properties in the absence or presence of fluoride anion were investigated. The results show that TDP not only exhibits large two-photon absorption (2PA) cross section (δ) up to 1030 GM and strong red two-photon excitation fluorescence, but also could operate as one- and two-photon chemosensor selectively for fluoride anion. Interestingly, it is observed that both the detection sensitivity of TDP to fluoride anion and the ratiometric change in emission spectra upon two-photon induced by fluoride anion are higher than one-photon excitation, implying that TDP could serve better as two-photon fluorescence sensor for fluoride anion. The large δ and red-shifted 2PA band promoted by triphenylamine end-group enable TDP to sense fluoride anion under longer excitation wavelength and lower input laser power.

Benzimidazole-Based Ratiometric Two-Photon Fluorescent Probes for Acidic pH in Live Cells and Tissues

Many aspects of cell metabolism are controlled by acidic pH. We report a new family of small molecule and ratiometric two photon (TP) probes derived from benzimidazole (BH1-3 and BH1L) for monitoring acidic pH values. These probes are characterized by a strong two-photon excited fluorescence, a marked blue-to-green emission color change in response to pH, pKa values ranging from 4.9 to 6.1, a distinctive isoemissive point, negligible cytotoxicity, and high photostability, thereby allowing quantitative analysis of acidic pH. Moreover, we show that BH1L optimized as a lysosomal-targeted probe allows for direct, real-time estimation of the pH values inside lysosomal compartments in live cells as well as in living mouse brain tissues through the use of two-photon microscopy. These findings demonstrate that these probes will find useful applications in biomedical research.

Synthesis, crystal structures, two-photon absorption and biological imaging application of two novel bent-shaped pyrimidine derivatives

Abstract Two novel bent-shaped D–π–A–π–D chromophore molecules, 2-pyrazolyl-4,6-bis (4-N,N-ethylaminostyryl)pyrimidine (Z1) and 2-imidazolyl-4,6 –bis- (4-N,N- ethylaminostyryl) pyrimidine (Z2), were synthesized and fully characterized. It was found that the both bent-shaped chromophore molecules crystallize in the orthorhombic system, space group of P212121. The photophysical properties and the connections between structure and properties of the chromophores were investigated both experimentally and theoretically. It was revealed that both absorption and single-photon excited fluorescence (SPEF) spectra show remarkable bathochromic shifts with increasing polarity of the solvents. The two chromophores have strong two-photon excited fluorescence and large two-photon absorption cross-sections in the near-infrared range. Additionally, two-photon microscopy (TPM) fluorescent imaging of BEL-7402 cells labeled with the Z1 and Z2 revealed their potential application as a biological fluorescent probe.

Synthesis and photophysical properties of two novel branched pyridinium inner salt dyes

Two new branched pyridinium inner salt dyes, 2,6-di[2-( N -ethyl-carbazole-3-yl)vinyl]- N -(3-sulfonatepropyl)pyridinium, inner salt ( 2 ) and 2,4,6-tri[2-( N -ethyl-carbazole-3-yl)vinyl]- N -(3-sulfonatepropyl)pyridinium, inner salt ( 3 ), with N -ethyl-carbazolyl as electron donor group and pyridine cation as electron acceptor group have been synthesized. Their single- and two-photon related fluorescence properties in various solvents have also been examined. These dyes exhibit stronger fluorescence emission relative to the corresponding linear counterpart and common pyridinium dyes with discrete cation and anion components. Three-branched organic dye 3 has a high fluorescence quantum yield as 0.12 and exhibits strong orange two-photon excited fluorescence at 608 nm in THF with large two-photon absorption cross-section being 606 GM. Two new branched pyridinium inner salt dyes with N -ethyl-carbazolyl as electron donor group have been synthesized. Three-branched organic dye has a high fluorescence quantum yield as 0.12 and exhibits strong orange two-photon excited fluorescence at 608 nm in THF with two-photon absorption cross-section being 606 GM. ► Two new branched pyridinium inner salt dyes were synthesized. ► The dyes exhibit strong yellow or orange fluorescence emission. ► Three-branched organic dye exhibits strong orange two-photon excited fluorescence in THF.

Fusing three perylenebisimide branches and a truxene core into a star-shaped chromophore with strong two-photon excited fluorescence and high photostability

A novel star-shaped chromophore, Tr-PBI, was constructed by fusing three perylenebisimide branches and a truxene core. Tr-PBI exhibits high photostability and excellent two-photon properties: the maximum of δ(TPA) is 11,000 GM at 990 nm and fluorescence quantum efficiency Φ is 0.40 in THF.

Combined Nonlinear and Femtosecond Confocal Laser-Scanning Microscopy of Rabbit Corneas after Photochemical Cross-Linking

Photochemical cross-linking of corneal stromal collagen using riboflavin and ultraviolet irradiation is an evolving treatment for keratoconus. The purpose of the present study was to investigate the wound-healing process in rabbit corneas after cross-linking. Photochemical cross-linking was performed according to a standard protocol on the right eyes of eight male New Zealand White rabbits; the left eyes served as controls. Untreated controls and cross-linked rabbit corneas were imaged 3 days, 6 days, and 6 weeks after treatment using a customized setup for three-dimensional nonlinear microscopy and confocal laser-scanning microscopy of reflected femtosecond light (fs-CLSM). The combination of fs-CLSM in reflective mode and two-photon-excited fluorescence permitted differentiation of the following zones in the lamina propria of treated corneas 3 and 6 days after cross-linking: (1) an anterior zone with postapoptotic keratocyte debris, visible only on fs-CLSM in reflective mode; (2) a posterior zone with activated keratocytes with strong autofluorescence; and (3) surviving or restored keratocytes with moderate autofluorescence beyond the intermediate zone. Repopulation with normal keratocytes was achieved by 6 weeks. Bi-directional, second-harmonic generation (SHG) imaging showed no global differences in the fiber orientation and lamellar structure of stromal collagen at any time point. A relatively strong additional two-photon excited fluorescence occurred in the treated corneas with a diffuse three-dimensional spatial distribution. This combination of imaging modalities has the potential to become a new clinical instrument capable of visualizing corneal changes at the cellular and extracellular level.

Two-photon excited fluorescence properties and anions recognition of a trivalent organoboron compound

A trivalent organoboron compound with diethylamino as an electron donor group and dimesitylboryl as an electron acceptor group has been synthesized. Its single- and two-photon excited fluorescence properties have been examined. Pumped by 820 nm laser pulses in femtosecond regime, it showed strong two-photon excited fluorescence at 527 nm in toluene with two-photon absorption cross-section being 859GM. The compound has been found to be a sensitive chemosensor for fluoride and cyanide anions in THF solutions.

Recognition of serous ovarian tumors in human samples by multimodal nonlinear optical microscopy

We used a multimodal nonlinear optics microscopy, specifically two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG∕THG) microscopies, to observe pathological conditions of ovarian tissues obtained from human samples. We show that strong TPEF + SHG + THG signals can be obtained in fixed samples stained with hematoxylin and eosin (H&E) stored for a very long time, and that H&E staining enhanced the THG signal. We then used the multimodal TPEF-SHG-THG microscopies in a stored file of H&E stained samples of human ovarian cancer to obtain complementary information about the epithelium∕stromal interface, such as the transformation of epithelium surface (THG) and the overall fibrillary tissue architecture (SHG). This multicontrast nonlinear optics microscopy is able to not only differentiate between cancerous and healthy tissue, but can also distinguish between normal, benign, borderline, and malignant specimens according to their collagen disposition and compression levels within the extracellular matrix. The dimensions of the layers of epithelia can also be measured precisely and automatically. Our data demonstrate that optical techniques can detect pathological changes associated with ovarian cancer.