An Activatable NIR-II Fluorescent Reporter for In Vivo Imaging of Amyloid-β Plaques

Fluorescence imaging in the second near-infrared (NIR-II) window holds great promise for in vivo visualization of amyloid-β (Aβ) pathology, which can facilitate characterization and deep understanding of Alzheimer's disease (AD); however, it has been rarely exploited. Herein, we report the development of NIR-II fluorescent reporters with a donor-π-acceptor (D-π-A) architecture for specific detection of Aβ plaques in AD-model mice. Among all the designed probes, DMP2 exhibits the highest affinity to Aβ fibrils and can specifically activate its NIR-II fluorescence after binding to Aβ fibrils via suppressed twisted intramolecular charge transfer (TICT) effect. With suitable lipophilicity for ideal blood–brain barrier (BBB) penetrability and deep-tissue penetration of NIR-II fluorescence, DMP2 possesses specific detection of Aβ plaques in in vivo AD-model mice. Thus, this study presents a potential agent for non-invasive imaging of Aβ plaques and deep deciphering of AD progression.     

Rational Design of a Robust Fluorescent Probe for the Detection of Endogenous Carbon Monoxide in Living Zebrafish Embryos and Mouse Tissue

Carbon monoxide (CO) is one of the most important gaseous signal molecules in biological systems. However, the investigation of the functions of CO in living organisms is restricted by the lack of functional molecular tools. To address this critical challenge, we present herein the rational design, synthesis, and in vivo imaging studies of a powerful two‐photon excited near‐infrared fluorescent probe ( 1‐Ac ) for endogenous CO monitoring. The advantageous features of the new probe include high stability, low background fluorescence, large fluorescence enhancement, high sensitivity, and two‐photon excitation with emission in the near‐infrared region. Significantly, these merits of the probe enable the tracking of endogenous CO in zebrafish embryos and mouse tissues for the first time.     

A Small‐Molecule Two‐Photon Probe for Nitric Oxide in Living Tissues

Two‐photon microscopy (TPM) has become an indispensable tool in the study of biology and medicine due to the capability of this method for molecular imaging deep inside intact tissues. For the maximum utilization of TPM, a variety of two‐photon (TP) probes for specific applications are needed. In this article, we report a small‐molecule TP probe (ANO1) for nitric oxide (NO) that shows a rapid and specific NO response, a 68‐fold fluorescence enhancement in response to NO, and a maximum TP‐action cross‐section of 170 GM (GM: 10^?50 cm⁴ photon^?1) upon reaction with excess NO. This probe can be easily loaded into cells and tissues and can real‐time monitor NO in living tissues at 100–180 μm depth for longer than 1200 s through the use of TPM, with minimum interference from other biologically relevant species.     

Photobleaching in Two‐Photon Scanning Fluorescence Correlation Spectroscopy

Two‐photon excitation in fluorescence correlation spectroscopy (FCS) is often preferred to one‐photon excitation because of reduced bulk photobleaching and photodamage, and deeper penetration into scattering media, such as thick biological specimens. Two‐photon FCS, however, suffers from lower signal‐to‐noise ratios which are directly related to the lower molecular brightness achieved. We compare standard FCS with a fixed measurement volume with scanning FCS, where the measurement volume is scanned along a circular path. The experimental results show that photobleaching is the dominant cause of the effects observed at the high excitation powers necessary for good signal‐to‐noise ratios. Theoretical calculations assuming a nonuniform excitation intensity profile, and using the concept of generalized volume contrast, provide an explanation for the photobleaching effects commonly observed in two‐photon FCS at high excitation intensities, without having to assume optical saturation. Scanning alleviates these effects by spreading the photobleaching dose over a larger area, thereby reducing the depletion of fluorescent molecules in the measurement volume. These results, which facilitate understanding of the photobleaching in FCS and of the positive effects of scanning, are particularly important in studies involving the autocorrelation amplitude g(0), such as concentration measurements or binding studies using fluorescence cross‐correlation between two labeled species.     

Two‐Photon Fluorescent Probes for Metal Ions

Two‐photon microscopy (TPM) has become an indispensible tool in biology and medicine owing to the capability of imaging the intact tissue for a long period of time. To make it a versatile tool in biology, a variety of two‐photon probes for specific applications are needed. In this context, many research groups are developing two‐photon probes for various applications. In this Focus Review, we summarize recent results on model studies and selected examples of two‐photon probes that can detect intracellular free metal ions in live cells and tissues to provide a guideline for the design of useful two‐photon probes for various in vivo imaging applications.     

Synthesis and two‐photon absorption properties of conjugated polymers with N‐arylpyrrole as conjugated bridge and isolation moieties

Three novel conjugated polymers with N‐arylpyrrole as the conjugated bridge were designed and synthesized, which emitted strong one‐ or two‐photon excitation fluorescence in dilute tetrahydrofuran (THF) solution with high quantum yields. The maximal two‐photon absorption (TPA) cross‐sections of the polymers, measured by the two‐photon‐induced fluorescence method using femtosecond laser pulses in THF, were 752, 1114, and 1869 GM, respectively, indicating that the insertion of electron‐donating or electron‐withdrawing moieties into the polymer backbone could benefit to the increase of the TPA cross‐section. Their large TPA cross‐sections, coupled with the relatively high emission quantum yields, made these conjugated polymers attractive for practical applications, especially two‐photon excited fluorescence. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

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.     

Mini‐Sized Carbon Nitride Nanosheets with Double Excitation‐ and pH‐Dependent Fluorescence Behaviors for Two‐Photon Cell Imaging

Synthesis of mini‐sized carbon nitride nanosheets (CNNSs) by traditional methods remains a challenge. Herein, size‐tunable and uniform mini‐sized CNNSs are synthesized by hydrothermal carbonization of a single polyethyleneimine (PEI) precursor. The as‐obtained mini‐sized CNNSs possess uniform size, good hydrophilicity and abundant nitrogen active sites, which not only exhibit double excitation‐ and pH‐dependent fluorescence behaviors, but also two‐photon excitation fluorescence. áThe resulting CNNSs display low toxicity and can be efficiently delivered into live cells for two‐photon fluorescence imaging, offering great potential as fluorescence probes in biochemical applications.     

Detection of nickel in fish organs with a two-photon fluorescent probe

Molecular imaging by two‐photon microscopy (TPM) has become indispensable to the study of biology/medicine owing to its capability of imaging deep inside intact tissues. To make TPM a more‐versatile tool, a large variety of two‐photon probes are needed. Herein, we report a new two‐photon fluorescent probe (ANi2) that can be excited by 750 nm femtosecond pulses and detect Ni²⁺ ions in fresh fish organs at 90–175 μm depth without interference from the pH value or from other biologically relevant species through the use of TPM. TPM images of fish organs labeled with ANi2 revealed that Ni²⁺ ions accumulate in fish organs in the order: kidney > heart > gill ≥ liver. Moreover, a linear relationship was found between the two‐photon‐excited fluorescence (TPEF) and the inductively coupled plasma mass spectrometry intensities (ICP‐MS), thereby allowing the quantitative measurement of Ni²⁺ ions in live tissue.     

Excited State Intramolecular Proton Transfer and Photophysics of a New Fluorenyl Two‐Photon Fluorescent Probe

The steady‐state photophysical, NMR, and two‐photon absorption (2PA) properties of a new fluorene derivative ( 1 ) containing the 2‐(2′‐hydroxyphenyl)benzothiazole (HBT) terminal construct is investigated for use as a fluorescence probe in bioimaging. A comprehensive analysis of the linear spectral properties reveals inter‐ and intramolecular hydrogen bonding and excited state intramolecular proton transfer (ESIPT) processes in the HBT substituent. A specific electronic model with a double minimum potential energy surface is consistent with the observed spectral properties. The 2PA spectra are obtained using a standard two‐photon induced fluorescence method with a femtosecond kHz laser system, affording a maximum 2PA cross section of ～600 GM, a sufficiently high value for two‐photon fluorescence imaging. No dependence of two‐photon absorption efficiency on solvent properties and hydrogen bonding in the HBT substituent is observed. The potential use of this fluorenyl probe in bioimaging is demonstrated via one‐ and two‐photon fluorescence imaging of COS‐7 cells.     

Synthesis and Nonlinear Optical Properties of a New A-π-A Two-Photon Compound Utilizing Dibenzothiophene as Center

A new two‐photon material, 3E,6E‐bis(2‐pyrid‐4′‐ylvinyl)dibenzothiophene (BPVDBT), has been firstly synthesized by an efficient Pd‐catalyzed Heck coupling route. The single‐ and two‐photon fluorescence, quantum yields, lifetimes, solvent effects of the chromophore were studied in detail and the compound exhibited solvent‐sensitivity. The fluorescence intensity (I_out) and input excitation intensity (I_in) can fit in well with the quadratic parabolas, which indicates that the up‐converted fluorescence was induced by the two‐photon absorption (TPA). TPA cross‐section of BPVDBT has been measured using the two‐photon‐induced fluorescence method, whose value is 14.24×10^?50 cm⁴·s·photon^?1·molecule^?1 at 750 nm. The experimental results confirm that BPVDBT is a good two‐photon absorbing chromophore with an A‐π‐A type.     

Water‐Soluble Triarylborane Chromophores for One‐ and Two‐Photon Excited Fluorescence Imaging of Mitochondria in Cells

Three water‐soluble tetracationic quadrupolar chromophores comprising two three‐coordinate boron π‐acceptor groups bridged by thiophene‐containing moieties were synthesised for biological imaging applications. Compound 3 containing the bulkier 5‐(3,5‐Me₂C₆H₂)‐2,2′‐(C₄H₂S)₂‐5′‐(3,5‐Me₂C₆H₂) bridge is stable over a long period of time, exhibits a high fluorescence quantum yield and strong one‐ and two‐photon absorption (TPA), and has a TPA cross section of 268 GM at 800 nm in water. Confocal laser scanning fluorescence microscopy studies in live cells indicated localisation of the chromophore at the mitochondria; moreover, cytotoxicity measurements proved biocompatibility. Thus, chromophore 3 has excellent potential for one‐ and two‐photon‐excited fluorescence imaging of mitochondrial function in cells.     

Readily Accessible and Predictable Naphthalene‐Based Two‐Photon Fluorophore with Full Visible‐Color Coverage

Herein we report 22 acedan‐derived, two‐photon fluorophores with synthetic feasibility and full coverage of visible wavelength emission. The emission wavelengths were predicted by computational analysis, which enabled us to visualize multicolor images by two‐photon excitation with single wavelength, and to design a turn‐on, two‐photon fluorescence sensor for endogenous H₂O₂ in Raw 264.7 macrophage and rat brain hippocampus ex vivo.     

Energy Transfer in Aminonaphthalimide‐Boron‐Dipyrromethene (BODIPY) Dyads upon One‐ and Two‐Photon Excitation: Applications for Cellular Imaging

Aminonaphthalimide–BODIPY energy transfer cassettes were found to show very fast (k_EET≈10¹⁰–10¹¹ s^?1) and efficient BODIPY fluorescence sensitization. This was observed upon one‐ and two‐photon excitation, which extends the application range of the investigated bichromophoric dyads in terms of accessible excitation wavelengths. In comparison with the direct excitation of the BODIPY chromophore, the two‐photon absorption cross‐section δ of the dyads is significantly incremented by the presence of the aminonaphthalimide donor [δ≈10 GM for the BODIPY versus 19–26 GM in the dyad at λ_exc=840 nm; 1 GM (Goeppert–Mayer unit)=10^?50 cm⁴ s molecule^?1 photon^?1]. The electronic decoupling of the donor and acceptor, which is a precondition for the energy transfer cassette concept, was demonstrated by time‐dependent density functional theory calculations. The applicability of the new probes in the one‐ and two‐photon excitation mode was demonstrated in a proof‐of‐principle approach in the fluorescence imaging of HeLa cells. To the best of our knowledge, this is the first demonstration of the merging of multiphoton excitation with the energy transfer cassette concept for a BODIPY‐containing dyad.     

Multilayer Microcapsules for FRET Analysis and Two‐Photon‐Activated Photodynamic Therapy

Microcapsules obtained by layer‐by‐layer assembly provide a good platform for biological analysis owing to their component diversity, multiple binding sites, and controllable wall thickness. Herein, different assembly species were obtained from two‐photon dyes and traditional photosensitizers, and further assembled into microcapsules. Fluorescence resonance energy transfer (FRET) was shown to occur between the two‐photon dyes and photosensitizers. Confocal laser scanning microscopy (CLSM) with one‐ and two‐photon lasers, fluorescence lifetime imaging microscopy (FLIM), and time‐resolved fluorescence spectroscopy were used to analyze the FRET effects in the microcapsules. The FRET efficiency could easily be controlled through changing the assembly sequence. Furthermore, the capsules are phototoxic upon one‐ or two‐photon excitation. These materials are thus expected to be applicable in two‐photon‐activated photodynamic therapy for deep‐tissue treatment.     

Iridium(III) Anthraquinone Complexes as Two‐Photon Phosphorescence Probes for Mitochondria Imaging and Tracking under Hypoxia

In the present study, four mitochondria‐specific and two‐photon phosphorescence iridium(III) complexes, Ir1 – Ir4 , were developed for mitochondria imaging in hypoxic tumor cells. The iridium(III) complex has two anthraquinone groups that are hypoxia‐sensitive moieties. The phosphorescence of the iridium(III) complex was quenched by the functions of the intramolecular quinone unit, and it was restored through two‐electron bioreduction under hypoxia. When the probes were reduced by reductase to hydroquinone derivative products under hypoxia, a significant enhancement in phosphorescence intensity was observed under one‐ (λ=405 nm) and two‐photon (λ=720 nm) excitation, with a two‐photon absorption cross section of 76–153 GM at λ=720 nm. More importantly, these probes possessed excellent specificity for mitochondria, which allowed imaging and tracking of the mitochondrial morphological changes in a hypoxic environment over a long period of time. Moreover, the probes can visualize hypoxic mitochondria in 3D multicellular spheroids and living zebrafish through two‐photon phosphorescence imaging.     

N‐Heterocyclic Carbene Boranes as Reactive Oxygen Species‐Responsive Materials: Application to the Two‐Photon Imaging of Hypochlorous Acid in Living Cells and Tissues

N‐Heterocyclic carbene (NHC) boranes undergo oxidative hydrolysis to give imidazolium salts with excellent kinetic selectivity for HOCl over other reactive oxygen species (ROS), including peroxides and peroxynitrite. Selectivity for HOCl results from the electrophilic oxidation mechanism of NHC boranes, which stands in contrast to the nucleophilic oxidation mechanism of arylboronic acids with ROS. The change in polarity that accompanies the conversion of NHC boranes to imidazolium salts can control the formation of emissive excimers, forming the basis for the design of the first fluorescence probe for ROS based on the oxidation of B?H bonds. Two‐photon microscope (TPM) ratiometric imaging of HOCl in living cells and tissues is demonstrated.     

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.     

A Dipolar Anthracene Dye: Synthesis,Optical Properties and Two‐photon Tissue Imaging

Two‐photon microscopy is a powerful tool for studying biological systems. In search of novel two‐photon absorbing dyes for bioimaging, we synthesized a new anthracene‐based dipolar dye (anthradan) and evaluated its two‐photon absorbing and imaging properties. The new anthradan, 9,10‐bis(o‐dimethoxy‐phenyl)‐anthradan, absorbs and emits at longer wavelengths than acedan, a well‐known two‐photon absorbing dye. It is also stable under two‐photon excitation conditions and biocompatible, and thus used for two‐photon imaging of mouse organ tissues to show bright, near‐red fluorescence along with negligible autofluorescence. Such an anthradan thus holds promise as a new class of two‐photon absorbing dyes for the development of fluorescent probes and tags for biological systems.     

Semiconducting Polymer Dots with Dual‐Enhanced NIR‐IIa Fluorescence for Through‐Skull Mouse‐Brain Imaging

Fluorescence probes in the NIR‐IIa region show drastically improved imaging owing to the reduced photon scattering and autofluorescence in biological tissues. Now, NIR‐IIa polymer dots (Pdots) are developed with a dual fluorescence enhancement mechanism. First, the aggregation induced emission of phenothiazine was used to reduce the nonradiative decay pathways of the polymers in condensed states. Second, fluorescence quenching was minimized by different levels of steric hindrance to further boost the fluorescence. The resulting Pdots displayed a fluorescence QY of ca. 1.7 % in aqueous solution, suggesting an enhancement of ca. 21 times in comparison with the original polymer in tetrahydrofuran (THF) solution. Small‐animal imaging by using the NIR‐IIa Pdots exhibited a remarkable improvement in penetration depth and signal to background ratio, as confirmed by through‐skull and through‐scalp fluorescent imaging of the cerebral vasculature of live mice.