Two-photon Excited Fluorescence Resonance Energy Research Articles

A NIR-triggered gatekeeper of supramolecular conjugated unimicelles with two-photon absorption for controlled drug release.

A near-infrared (NIR)-sensitive gated assembly of supramolecular conjugated unimicelles based on robust host-guest recognition between a β-cyclodextrin-grafted hyperbranched conjugated polymer and azobenzene-functionalized poly(ethylene glycol) was constructed. Utilized as a drug carrier, these unimicelles exhibited controlled drug release through the NIR-triggered photoisomerization of azobenzene in cancer cells via a two-photon excited fluorescence resonance energy transfer (TP-FRET) approach, leading to efficient cancer therapy.

A Ratiometric Two-Photon Fluorescent Cysteine Probe with Well-Resolved Dual Emissions Based on Intramolecular Charge Transfer-Mediated Two-Photon-FRET Integration Mechanism.

The development of an efficient ratiometric two-photon fluorescence imaging probe is crucial for in situ monitoring of biothiol cysteine (Cys) in biosystems, but the current reported intramolecular charge transfer (ICT)-based one suffers from serious overlap between the shifted emission bands. To address this issue, we herein for the first time constructed an ICT-mediated two-photon excited fluorescence resonance energy transfer (TP-FRET) system consisting of a two-photon fluorogen benzo[ h]chromene and a Cys-responsive benzoxadiazole-analogue dye. Different from a previous mechanism that utilized single two-photon fluorogen to acquire a ratiometric signal, ICT was used to switch on the TP-FRET process of the energy transfer dyad by eliciting an absorption shift of benzoxadiazole with Cys to modulate the spectral overlap level between benzo[ h]chromene emission and benzoxadiazole absorption, resulting in two well-separated emission signal changes with large emission wavelength shift (120 nm), fixed two-photon excitation maximum (750 nm), and significant variation in fluorescence ratio (over 36-fold). Therefore, it can be successfully employed to ratiometrically visualize Cys in HeLa cells and liver tissues. Importantly, this new ICT-mediated TP-FRET integration mechanism would be convenient for designing ratiometric two-photon fluorescent probes with two well-resolved emission spectra suitable for high resolution two-photon fluorescence bioimaging.

Combining Two-Photon-Activated Fluorescence Resonance Energy Transfer and Near-Infrared Photothermal Effect of Unimolecular Micelles for Enhanced Photodynamic Therapy.

Recent years have witnessed significant progress in the field of two-photon-activated photodynamic therapy (2P-PDT). However, the traditional photosensitizer (PS)-based 2P-PDT remains a critical challenge in clinics due to its low two-photon absorption (2PA) cross sections. Here, we propose that the therapeutic activity of current PSs can be enhanced through a combination of two-photon excited fluorescence resonance energy transfer (FRET) strategy and photothermal effect of near-infrared (NIR) light. A core-shell unimolecular micelle with a large two-photon-absorbing conjugated polymer core and thermoresponsive shell was constructed as a high two-photon light-harvesting material. After PSs were grafted onto the surface of a unimolecular micelle, the FRET process from the conjugated core to PSs could be readily switched "on" to kill cancer by the collapsed thermoresponsive shell due to the photothermal effect of NIR light. Such NIR-triggered FRET leads to an enhanced 2PA activity of the traditional PSs and, in turn, amplifies their cytotoxic singlet oxygen generation. Eventually, both in vitro and in vivo PDT efficiencies treated with the thermoresponsive micelles were dramatically enhanced under NIR light irradiation, as compared to pure PSs excited by traditional visible light. Such a facile and simple methodology for the enhancement of the photodynamic antitumor effect holds great promises for cancer therapy with further development.

Two-photon-excited fluorescence resonance energy transfer in an aqueous system of CdTe quantum dots and Rhodamine B

Two-photon excited fluorescence resonance energy transfer (FRET) between CdTe quantum dots with different emission peaks and Rhodamine B in aqueous solution are investigated both experimentally and theoretically. The photoluminescence and lifetime are measured using a time-resolved fluorescence test system. The two-photon excited FRET efficiency is found to increase as the degree of spectral overlap of the emission spectrum of CdTe and the absorption spectrum of Rhodamine B increases, which is due to the increase of Forster radius of the sample. Moreover, FRET efficiency increases when the ratio of acceptor/donor concentration increases. The two-photon excited FRET efficiency was found to reach 40%.

Construction of a molecular beacon based on two-photon excited fluorescence resonance energy transfer with quantum dot as donor

A new molecular beacon (MB) driven by two-photon excitation (TPE) using quantum dots as energy donor is constructed, which provides reduced direct excitation of acceptor and is free of interferences from autofluorescence or scattering light in a complicated biological matrix.

Homogeneous Competitive Hybridization Assay Based on Two-Photon Excitation Fluorescence Resonance Energy Transfer

Recently, we have successfully developed a two-photon excitation fluorescence resonance energy transfer (TPE-FRET)-based homogeneous immunoassay using two-photon excitable small organic molecule as the energy donor. In the present work, the newly emerging TPE-FRET technique was extended to the determination of oligonucleotide. A new TPE molecule with favorable two-photon action cross section was synthesized [2-(2,5-bis(4-(dimethylamino)styryl)-1H-pyrrol-1-yl)acetic acid, abbreviated as TP-COOH], with the tagged reactive carboxyl group allowing facile conjugation with streptavidin (SA). Employing the TP-COOH molecule as energy donor and black hole quencher 1 (BHQ-1) as acceptor, a TPE-FRET-based homogeneous competitive hybridization model was constructed via a biotin-streptavidin bridge. Through the hybridization between a biotinylated single-stranded DNA (ssDNA) and a BHQ-1-linked ssDNA, and the subsequent capture of the as-formed hybrid by TP-COOH labeled SA, the donor fluorescence was quenched due to the FRET between TP-COOH and BHQ-1. Upon the competition between a target ssDNA and the quencher-linked ssDNA toward the biotinylated oligonucleotide, the donor fluorescence was recovered in a target-dependent manner. Good linearity was obtained with the target oligonucleotide ranging from 0.08 to 1.52 microM. The method was applied to spiked serum and urine samples with satisfying recoveries obtained. The results of this work verified the applicability of TPE-FRET technique in hybridization assay and confirmed the advantages of TPE-FRET in complicated matrix.

Enhancement of two-photon absorption cross section and singlet-oxygen generation in porphyrin-cored star polymers

We report a newly synthesized polymer of a star-shaped porphyrin compound (TPA-FxP) with four oligofluorene arms at its meso positions with the pronounced enhancement of the two-photon properties and the generation of singlet oxygen by utilizing the two-photon excited fluorescence resonance energy transfer. The steady-state spectra and transient triplet-triplet absorption spectra give evidence that the enhanced two-photon absorption cross section results from not only the through-space energy transfer (Forster) but also the through-bond energy transfer between conjugated peripheral oligofluorene arms and the porphyrin core. The two-photon absorption cross section at 780 nm up to 3360 GM (1 GM = 10−50 cm4·s/photon) of TPA-FxP was obtained, which is comparable to the highest values reported from other similar chemically modified porphyrin core compounds. Furthermore, the enhanced production of singlet oxygen under two-photon absorption conditions is also reported.

Homogeneous Immunoassay Based on Two-Photon Excitation Fluorescence Resonance Energy Transfer

A two-photon excitable small organic molecule (abbreviated as TP-NH 2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared, which emitted fluorescence in the visible region upon excitation at 800 nm. Using the TP-NH 2 molecule as an energy donor, a two-photon excitation fluorescence resonance energy-transfer (TPE-FRET) based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-Cl, a dark quencher) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.

Two-Photon Excitation Fluorescence Resonance Energy Transfer with Small Organic Molecule as Energy Donor for Bioassay

A fluorescence resonance energy transfer (FRET) model using two-photon excitable small organic molecule DMAHAS as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-Cl. Binding of DABS-Cl labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light, the coexcitation of the energy acceptor with the donor. And such small molecules also show advantages over inorganic up-converting particles that also give anti-Stokes photoluminescence and have been applied as FRET donor recently. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor for FRET-based bioassays.

Enhanced Two‐Photon Excitation Fluorescence by Fluorescence Resonance Energy Transfer Using Conjugated Polymers

A two-photon excitation fluorescence resonance energy transfer scheme, in a system consisting of a cationic conjugated polymer, dsDNA, and a DNA intercalator, is reported. The conjugated polymer acts as two-photon excitation light-harvesting complex (see figure). The two-photon excitation fluorescence of the intercalator is found to be enhanced by a factor of over 35 upon addition of conjugated polymers. The obtained results may have profound implications in two-photon imaging and phototherapy.

Characterization of two‐photon excitation fluorescence lifetime imaging microscopy for protein localization

Two-photon excitation fluorescence resonance energy transfer (2P-FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P-FRET imaging have characteristic absorption and emission spectra that introduce spectral cross-talk (bleed-through) in the FRET signal that should be removed in the 2P-FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P-FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein-protein interactions in living cells and tissues. 2P-FRET-FLIM was used to study the dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P-FRET-FLIM imaging system with the Bio-Rad Radiance2100 confocal/multiphoton microscopy system.

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein α (C/EBPα). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency ( E) and the distance ( r) between donor and acceptor molecules by 38 and 9%, respectively.