Two-photon Fluorescence Bioimaging Research Articles

Efficient Near-Infrared Fluorophores Based on Cyanostyrene Derivatives for Two-Photon Fluorescence Bioimaging.

Organic fluorescent materials with red/near-infrared (NIR) emission are highly promising for use in biotechnology due to their exceptional advantages. However, traditional red/NIR fluorophores often exhibit weak emission at high concentrations or in an aggregated state due to the aggregate-caused quenching effect, which severely limits their applicability in biological imaging. To address this challenge, we developed a series of cyanostyrene derivatives with aggregation-induced emission characteristics, including 2,3-Bis-(4-styryl-phenyl)-but-2-enedinitrile (DPB), 2,3-Bis-{4-[2-(4-methoxy- phenyl)-vinyl]-phenyl}-but-2-enedinitrile (DOB), 2,3-Bis-{4-[2-(4-diphenylamino- phenyl)-vinyl]-phenyl}-but-2-enedinitrile (DTB), and 2,3-Bis-[4-(2-{4-[phenyl- (4-triphenylvinyl-phenyl)-amino]-phenyl}-vinyl)- phenyl]-but-2-enedinitrile (DTTB). Notably, these compounds exhibited intense solid state fluorescence owing to AIE effect, especially DTTB shows NIR emission with high solid state quantum efficiency (712 nm, ΦF=14.2 %). Then we prepared DTTB@PS-PEG NPs nanoparticles by encapsulating DTTB with the amphiphilic polymer polystyrene-polyethylene glycol (PS-PEG). Importantly, DTTB@PS-PEG NPs exhibited highly efficient NIR luminescence (ΦF=28.7 %) and a large two-photon absorption cross-section (1900 GM) under 800 nm laser excitation. The bright two-photon fluorescence of DTTB@PS-PEG indicated that it can be a highly promising candidate for two-photon fluorescence probe. Therefore, this work provides valuable insights for the design of highly efficient and NIR-emitting two-photon fluorescent probes.

Structure-activity relationships of aniline-based squaraines for distinguishable staining and bright two-photon fluorescence bioimaging in plant cells.

An organelle-selective vision provides insights into the physiological response of plants and crops to environmental stresses in sustainable agriculture ecosystems. Biological applications often require two-photon excited fluorophores with low phototoxicity, high brightness, deep penetration, and tuneable cell entry. We obtained three aniline-based squaraines (SQs) tuned from hydrophobic to hydrophilic characteristics by modifying terminal pendant groups and substituents, and investigated their steady-state absorption and far-red-emitting fluorescence properties. The SQs exhibited two-photon absorption (2PA) ranging from 750 to 870 nm within the first biological spectral window; their structure-property relationships, corresponding to the 2PA cross sections (δ2PA), and structure differences were demonstrated. The maximum δ2PA value was ∼1220 GM at 800 nm for hydrophilic SQ3. Distinct biological staining efficiency and selective SQ bioimaging were evaluated utilizing the onion epidermal cell model. Contrary to the hydrophobic SQ1 results in the onion epidermal cell wall, amphiphilic SQ2 tagged the vacuole and nucleus and SQ3 tagged the vacuole. Distinguishable staining profiles in the roots and leaves were achieved. We believe that this study is the first to demonstrate distinct visualisation efficiency induced by the structure differences of two-photon excited SQs. Our results can help establish the versatile roles of novel near-infrared-emitting SQs in biological applications.

Achieving two-photon fluorescence bioimaging and photodynamic therapy for D–A conjugated polymers through manipulating twisted intramolecular charge transfer

Two-photon fluorescence bioimaging holds immense promise for deep tissue imaging.

Two-photon fluorescence and MR bio-imaging of endogenous H2O2 in the tumor microenvironment using a dual-mode nanoprobe.

The dual-mode bio-imaging nanoprobe TP-CQDs@MnO2, based on two-photon carbon quantum dots and MnO2, has been developed for the two-photon fluorescence and MR imaging of endogenous H2O2 in the tumor microenvironment, and it achieved high selectivity, a great signal-to-noise ratio, a limit of detection (LOD) of 1.425 pM for H2O2, and a two-photon tissue penetration depth of 280 μm.

Multicationic AIEgens for unimolecular photodynamic theranostics and two-photon fluorescence bioimaging

This work reports multicationic AIEgens for unimolecular photodynamic theranostics, and an investigation of the relationship between their structures and activities, including PDT performance, 2-photon bioimaging, and self-reporting characteristics.

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.

Aggregation-Induced Emission Luminogen with Near-Infrared-II Excitation and Near-Infrared-I Emission for Ultradeep Intravital Two-Photon Microscopy

Currently, a serious problem obstructing the large-scale clinical applications of fluorescence technique is the shallow penetration depth. Two-photon fluorescence microscopic imaging with excitation in the longer-wavelength near-infrared (NIR) region (>1100 nm) and emission in the NIR-I region (650-950 nm) is a good choice to realize deep-tissue and high-resolution imaging. Here, we report ultradeep two-photon fluorescence bioimaging with 1300 nm NIR-II excitation and NIR-I emission (peak ∼810 nm) based on a NIR aggregation-induced emission luminogen (AIEgen). The crab-shaped AIEgen possesses a planar core structure and several twisting phenyl/naphthyl rotators, affording both high fluorescence quantum yield and efficient two-photon activity. The organic AIE dots show high stability, good biocompatibility, and a large two-photon absorption cross section of 1.22 × 103 GM. Under 1300 nm NIR-II excitation, in vivo two-photon fluorescence microscopic imaging helps to reconstruct the 3D vasculature with a high spatial resolution of sub-3.5 μm beyond the white matter (>840 μm) and even to the hippocampus (>960 μm) and visualize small vessels of ∼5 μm as deep as 1065 μm in mouse brain, which is among the largest penetration depths and best spatial resolution of in vivo two-photon imaging. Rational comparison with the AIE dots manifests that two-photon imaging outperforms the one-photon mode for high-resolution deep imaging. This work will inspire more sight and insight into the development of efficient NIR fluorophores for deep-tissue biomedical imaging.

Aggregation-Induced Emission Nanoparticles Encapsulated with PEGylated Nano Graphene Oxide and Their Applications in Two-Photon Fluorescence Bioimaging and Photodynamic Therapy in Vitro and in Vivo.

Aggregation-induced emission (AIE) nanoparticles have been shown promise for fluorescence bioimaging and photodynamic therapy due to the good combination of nanoparticles and organic dyes or photosensitizers. Among several kinds of AIE nanoparticles, those that are capsulated with nanographene oxides (NGO) are easy to make, size-tunable, and have proven to be very stable in deionized water. However, the stability in saline solution still needs improvement for further applications in chemical or biomedical fields, and the efficacy of photodynamic therapy using NGO-capsulate AIE photosensitizers has not been evaluated yet. Herein, we modified NGO with polyethylene glycol (PEG) to improve the stability of NGO-capsulated AIE nanoparticles in phosphate buffer saline. Furthermore, by combining this modification methodwith a dual-functional moleculewhich has both typical AIE property and photosensitizing ability, we performed both two-photon fluorescence bioimaging and photodynamic therapy in vitro and in vivo. Our work shows that AIE nanoparticles capsulated with PEGylated nanographene oxide can be a powerful tool for future bioimaging and photodynamic therapy applications.

Tumor-Targeted Graphitic Carbon Nitride Nanoassembly for Activatable Two-Photon Fluorescence Imaging.

Unique physicochemical characteristics of graphitic carbon nitride (g-CN) nanosheets suit them to be a useful tool for two-photon fluorescence bioimaging. Current g-CN nanosheets based imaging probes typically use the "always-on" design strategies, which may suffer from increased fluorescence background and limited contrast. To advance corresponding applications, g-CN nanosheets based activatable two-photon fluorescence probes remain to be explored. For the first time, we developed an activatable two-photon fluorescence probe, constructed from a nanoassembly of g-CN nanosheets and hyaluronic acid (HA)-gold nanoparticles (HA-AuNPs), for detection and imaging of hyaluronidase (HAase) in cancer cells. The deliberately introduced HA in our design not only functions as the buffering layer for stabilizing AuNPs and inducing corresponding self-assembly on g-CN nanosheets but also as a pilot for targeting HA receptors overexpressed on cancer cell surfaces. Our results show that the developed nanoassembly enables specific detection and activatable imaging of HAase in cancer cells and deep tissues, with superb signal-to-background ratio and high sensitivity. This nanoassembly can afford a promising platform for highly specific and sensitive imaging of HAase and for related cancer diagnosis.

Efficient Red/Near‐Infrared Fluorophores Based on Benzo[1,2‐b:4,5‐b′]dithiophene 1,1,5,5‐Tetraoxide for Targeted Photodynamic Therapy and In Vivo Two‐Photon Fluorescence Bioimaging

AbstractRed/near‐infrared dyes are highly demanded for biological applications but most of them are far from satisfactory. In this work, a series of red/near‐infrared fluorophores based on electron‐withdrawing benzo[1,2‐b:4,5‐b′]dithiophene 1,1,5,5‐tetraoxide (BDTO) are synthesized and characterized. They possess both aggregation‐induced emission, and hybridized local and charge‐transfer characteristics. Crystallographic, spectroscopic, electrochemical and computational results reveal that the oxidation of benzo[1,2‐b:4,5‐b′]dithiophene to BDTO can endow the fluorophores with greatly red‐shifted emission, enhanced emission efficiency, reduced energy levels, enlarged two‐photon absorption cross section, and increased reactive oxygen species generation efficiency. The nanoparticles fabricated with a near‐infrared fluorophore TPA‐BDTO show high photostability and biocompatibility with good performance in targeted photodynamic ablation of cancer cells and two‐photon fluorescence imaging of intravital mouse brain vasculature.

Ultrathin two-dimensional covalent organic framework nanoprobe for interference-resistant two-photon fluorescence bioimaging.

The complex environment of living organisms significantly challenges the selectivity of classic small-molecule fluorescent probes for bioimaging. Due to their predesigned topological structure and engineered internal pore surface, covalent organic frameworks (COFs) have the ability to filter out coexisting interference components and help to achieve accurate biosensing. Herein, we propose an effective interference-resistant strategy by creating a COF-based hybrid probe that combines the respective advantages of COFs and small-molecule probes. As a proof of concept, a two-photon fluorescent COF nanoprobe, namely TpASH-NPHS, is developed for targeting hydrogen sulfide (H2S) as a model analyte. TpASH-NPHS exhibits limited cytotoxicity, excellent photostability and long-term bioimaging capability. More importantly, compared with the small-molecule probe, TpASH-NPHS achieves accurate detection without the interference from intracellular enzymes. This allows us to monitor the levels of endogenous H2S in a mouse model of cirrhosis.

A facile and one-step ethanol-thermal synthesis of MoS2 quantum dots for two-photon fluorescence imaging.

Two-photon fluorescent (TPF) molybdenum disulfide quantum dots (MoS2 QDs) were synthesized through a facile and one-step solvothermal approach. The MoS2 QDs exhibit small size and high stability. Because of their low toxicity and TPF ability, the MoS2 QDs are successfully applied in two-photon fluorescence bio-imaging.

Near-infrared emissive carbon dots for two-photon fluorescence bioimaging.

Fluorescent probes with both excitation and emission in the near-infrared (NIR) region are highly attractive in the field of bioimaging. Herein, NIR emissive carbon dots (CDs, λmax = 683 nm) capable of excitation with a NIR femtosecond pulse laser (850 nm) are reported for the first time. The NIR CDs also hold a variety of superior features including excellent water dispersibility, narrow emission bands, and high quantum yields (QY = 16.8%). Further studies reveal that the emission of the NIR CDs derives predominantly from the surface molecular state mechanism. More interestingly, the NIR CDs are verified to have very low cytotoxicity and show capabilities for two-photon fluorescence bioimaging. Finally, the NIR CDs are shown to be easily modified with fluorescein isothiocyanate (FITC) and moved into living cells. These findings demonstrate that the as-prepared NIR CDs could not only be potentially employed for deep-tissue two-photon bioimaging, but also used as an effective carrier for delivery of drugs that can't (or hardly) enter into living cells directly.

Organelle-Specific Detection of Phosphatase Activities with Two-Photon Fluorogenic Probes in Cells and Tissues

Two-photon fluorescence microscopy (TPFM) provides key advantages over conventional fluorescence imaging techniques, namely, increased penetration depth, lower tissue autofluorescence and self-absorption, and reduced photodamage and photobleaching and therefore is particularly useful for imaging deep tissues and animals. Enzyme-detecting, small molecule probes provide powerful alternatives over conventional fluorescent protein (FP)-based methods in bioimaging, primarily due to their favorable photophysical properties, cell permeability, and chemical tractability. In this article, we report the first fluorogenic, small molecule reporter system (Y2/Y1) capable of imaging endogenous phosphatase activities in both live mammalian cells and Drosophila brains. The one- and two-photon excited photophysical properties of the system were thoroughly investigated, thus confirming the system was indeed a suitable Turn-ON fluorescence pair for TPFM. To our knowledge, this is the first enzyme reporting two-photon fluorescence bioimaging system which was designed exclusively from a centrosymmetric dye possessing desirable two-photon properties. By conjugation of our reporter system to different cell-penetrating peptides (CPPs), we were able to achieve organelle- and tumor cell-specific imaging of phosphatase activities with good spatial and temporal resolution. The diffusion problem typically associated with most small molecule imaging probes was effectively abrogated. We further demonstrated this novel two-photon system could be used for imaging endogenous phosphatase activities in Drosophila brains with a detection depth of >100 μm.

Folate receptor targeting silica nanoparticle probe for two-photon fluorescence bioimaging

Narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, were synthesized by hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol-OT micelles. The surface of the SiNPs were functionalized with folic acid, to specifically deliver the probe to folate receptor (FR) over-expressing Hela cells, making these folate two-photon dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing Hela cells and low FR expressing MG63 cells demonstrated specific cellular uptake of the functionalized nanoparticles. One-photon fluorescence microscopy (1PFM) imaging, 2PFM imaging, and two-photon fluorescence lifetime microscopy (2P-FLIM) imaging of Hela cells incubated with folate-modified two-photon dye-doped SiNPs were demonstrated.

Aggregation‐Enhanced Fluorescence in Organically Modified Silica Nanoparticles: A Novel Approach toward High‐Signal‐Output Nanoprobes for Two‐Photon Fluorescence Bioimaging

Phase-separated nanoaggregation of a highly loaded 9,10-bis[4'-(4''-aminostyryl)styryl]anthracene dye in organically modified silica (ORMOSIL) nanoparticles boosts intracellular two-photon fluorescence signal by virtue of enhanced two-photon absorption and emission, high chromophore number density in the nano-space, and noninvasive cellular uptake.

Two-photon fluorescence bioimaging with an all-semiconductor laser picosecond pulse source

We have demonstrated successful two-photon excitation fluorescence bioimaging using a high-power pulsed all-semiconductor laser. Toward this purpose, we developed a pulsed light source consisting of a mode-locked laser diode and a two-stage diode laser amplifier. This pulsed light source provided optical pulses of 5 ps duration and having a maximum peak power of over 100 W at a wavelength of 800 nm and a repetition frequency of 500 MHz.

Two-photon bioimaging utilizing supercontinuum light generated by a high-peak-power picosecond semiconductor laser source

We developed a novel scheme for two-photon fluorescence bioimaging. We generated supercontinuum (SC) light at wavelengths of 600 to 1200 nm with 774-nm light pulses from a compact turn-key semiconductor laser picosecond light pulse source that we developed. The supercontinuum light was sliced at around 1030- and 920-nm wavelengths and was amplified to kW-peak-power level using laboratory-made low-nonlinear-effects optical fiber amplifiers. We successfully demonstrated two-photon fluorescence bioimaging of mouse brain neurons containing green fluorescent protein (GFP).

Two-photon bioimaging with picosecond optical pulses from a semiconductor laser

Toward a practical light source for two-photon bioimaging, we have generated kilowatt peak power of 0.77 mum wavelength and 5 ps optical pulse via second-harmonic generation of the amplified output from a gain-switched 1.55 mum semiconductor laser. This compact scheme and stable optical-pulse-source has been successfully used for the two-photon fluorescence bioimaging of actin filaments in PtK2 cells.