A Strategy for Specific Fluorescence Imaging of Monoamine Oxidase A in Living Cells

Monoamine oxidase (MAO) has two isoforms, MAO‐A and MAO‐B, which show different functions, and thus selective fluorescence imaging is important for biological studies. Currently, however, specific detection of MAO‐A remains a great challenge. Herein, we report a new strategy for specific imaging of MAO‐A through the design of fluorogenic probes combining the characteristic structure of an inhibitor of the target enzyme along with propylamine as a recognition moiety. The high specificity of our representative probe is demonstrated by imaging MAO‐A in different live cells such as SH‐SY5Y (high levels of MAO‐A) and HepG2 (high levels of MAO‐B), and further validated by western blot analyses. The superior specificity of the probe may enable the accurate detection of MAO‐A in complex biosystems. Importantly, the use of the characteristic structure of an inhibitor, as demonstrated in this work, may serve as a general strategy to design specific recognition moieties for fluorogenic probes for enzymes.     

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.     

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.     

Harnessing Intramolecular Rotation To Enhance Two‐photon Imaging of Aβ Plaques through Minimizing Background Fluorescence

The aggregation of amyloid beta (Aβ) proteins in senile plaques is a critical event during the development of Alzheimer's disease, and the postmortem detection of Aβ‐rich proteinaceous deposits through fluorescent staining remains one of the most robust diagnostic tools. In animal models, fluorescence imaging can be employed to follow the progression of the disease, and among the different imaging methods, two‐photon microscopy (TPM) has emerged as one of the most powerful. To date, several near‐infrared‐emissive two‐photon dyes with a high affinity for Aβ fibrils have been developed, but there has often been a tradeoff between excellent two‐photon cross‐sections and large fluorescence signal‐to‐background ratios. In the current work, we introduced a twisted intramolecular charge state (TICT)‐based de‐excitation pathway, which results in a remarkable fluorescence increase of around 167‐fold in the presence of Aβ fibrils, while maintaining an excellent two‐photon cross section, thereby enabling high‐contrast ex vivo and in vivo TPM imaging. Overall, the results suggest that adopting TICT de‐excitation in two‐photon fluorophores may represent a general method to overcome the tradeoff between probe brightness and signal‐to‐background ratio.     

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.     

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.     

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.     

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.     

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.     

A Self‐Assembled Nanohybrid Composed of Fluorophore–Phenylamine Nanorods and Ag Nanocrystals: Energy Transfer,Wavelength Shift of Fluorescence and TPEF Applications for Live‐Cell Imaging

A fluorophore–phenylamine derivative ( L ) has been coupled with silver nanocrystals (NCs) to construct an L– Ag nanohybrid. Owing to synergic effects of the L and Ag components, the exciton–plasmon interactions between L and Ag increase the strength of the donor–acceptor interaction within the nanohybrid, a fact that results in an energy‐transfer process and further brings about a dramatic redshift of single‐photon absorption and fluorescence, and a decreased fluorescence FL lifetime. The coupling effect also leads to enhancement of a series of nonlinear optical properties, including two‐photon‐excited fluorescence (TPEF), two‐photon‐absorption (TPA) cross section (δ), two‐photon‐absorption coefficient (β), nonlinear refractive index (γ), and third order nonlinear optical susceptibility (χ⁽³⁾). The enhanced two‐photon fluorescence of the nanohybrid is proven to be potentially useful for two‐photon microscopy of live cells, such as HepG2. Moreover, cytotoxicity tests show that the low‐micromolar concentrations of the nanohybrid do not cause significant reduction in cell viability over a period of at least 24 h and should be safe for further biological studies.     

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.     

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     

Glycosylated Star‐Shaped Conjugated Oligomers for Targeted Two‐Photon Fluorescence Imaging

A glucopyranose functionalized star‐shaped oligomer, N‐tris{4,4′,4′′‐[(1E)‐2‐(2‐{(E)‐2‐[4‐(benzo[d]thiazol‐2‐yl)phenyl]vinyl}‐9,9‐bis(6‐2‐amido‐2‐deoxy‐1‐thio‐β‐D ‐glucopyranose‐hexyl)‐9H‐fluoren‐7‐yl)vinyl]phenyl}phenylamine (TVFVBN‐S‐NH₂), is synthesized for two‐photon fluorescence imaging. In water, TVFVBN‐S‐NH₂ self‐assembles into nanoparticles with an average diameter of ～49 nm and shows a fluorescence quantum yield of 0.21. Two‐photon fluorescence measurements reveal that TVFVBN‐S‐NH₂ has a two‐photon absorption cross‐section of ～1100 GM at 780 nm in water. The active amine group on the glucopyranose moiety allows further functionalization of TVFVBN‐S‐NH₂ with folic acid to yield TVFVBN‐S‐NH₂FA with similar optical and physical properties as those for TVFVBN‐S‐NH₂. Cellular imaging studies reveal that TVFVBN‐S‐NH₂FA has increased uptake by MCF‐7 cells relative to that for TVFVBN‐S‐NH₂, due to specific interactions between folic acid and folate receptors on the MCF‐7 cell membrane. This study demonstrates the effectiveness of glycosylation as a molecular engineering strategy to yield water‐soluble materials with a large two‐photon absorption (TPA) cross‐section for targeted cancer‐cell imaging.     

Solvent‐Switching Gelation and Orange–Red Emission of Ultrasmall Copper Nanoclusters

By tuning the Cu???Cu and hydrogen‐bonding interactions, the small cluster Cu₃L can be selectively synthesized to develop a stable and highly fluorescent material, as confirmed by matrix‐assisted laser desorption ionization–time of flight mass spectroscopy. Further characterizations, including absorbance spectroscopy, XPS, and XRD demonstrate the formation of tiny Cu nanoclusters (NCs). In water, the as‐prepared Cu NCs can exhibit high orange fluorescence via solution evaporation to eliminate hydrogen‐bonding, and in dimethylformamide, a strong orange fluorescent gel is obtained by solvent induction to enhance the Cu???Cu and hydrogen‐bonding interactions. More importantly, the Cu NCs in their substantial form exhibit nonlinear optical properties upon two‐photon excitation. These results will shed light on Cu and related cluster applications in two‐photon biological imaging, optical power limiting, and solar energy conversion.     

Covalent Organic Framework for Improving Near‐Infrared Light Induced Fluorescence Imaging through Two‐Photon Induction

Fluorescent materials exhibiting two‐photon induction (TPI) are used for nonlinear optics, bioimaging, and phototherapy. Polymerizations of molecular chromophores to form π‐conjugated structures were hindered by the lack of long‐range ordering in the structure and strong π–π stacking between the chromophores. Reported here is the rational design of a benzothiadiazole‐based covalent organic framework (COF) for promoting TPI and obtaining efficient two‐photon induced fluorescence emissions. Characterization and spectroscopic data revealed that the enhancement in TPI performance is attributed to the donor‐π‐acceptor‐π‐donor configuration and regular intervals of the chromophores, the large π‐conjugation domain, and the long‐range order of COF crystals. The crystalline structure of TPI‐COF attenuates the π–π stacking interactions between the layers, and overcomes aggregation‐caused emission quenching of the chromophores for improving near‐infrared two‐photon induced fluorescence imaging.     

Rational Design of Cyclometalated Iridium(III) Complexes for Three‐Photon Phosphorescence Bioimaging

Compared to 2PE (two‐photon excitation) microscopy, 3PE microscopy has superior spatial resolution, deeper tissue penetration, and less defocused interference. The design of suitable agents with a large Stokes shift, good three‐photon absorption (3PA), subcellular targeting, and fluorescence lifetime imaging (FLIM) properties, is challenging. Now, two Ir^III complexes (3PAIr1 and 3PAIr2) were developed as efficient three‐photon phosphorescence (3PP) agents. Calculations reveal that the introduction of a new group to the molecular scaffold confers a quadruple promotion in three‐photon transition probability. Confocal and lifetime imaging of mitochondria using Ir^III complexes as 3PP agents is shown. The complexes exhibit low working concentration (50 nm ), fast uptake (5 min), and low threshold for three‐photon excitation power (0.5 mW at 980 nm). The impressive tissue penetration depth (ca. 450 μm) allowed the 3D imaging and reconstruction of brain vasculature from a living specimen.     

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.     

A Small‐Molecule Probe for Selective Profiling and Imaging of Monoamine Oxidase B Activities in Models of Parkinson’s Disease

The design of the first dual‐purpose activity‐based probe of monoamine oxidase B (MAO‐B) is reported. This probe is highly selective towards MAO‐B, even at high MAO‐A expression levels, and could sensitively report endogenous MAO‐B activities by both in situ proteome profiling and live‐cell bioimaging. With a built‐in imaging module as part of the probe design, the probe was able to accomplish what all previously reported MAO‐B imaging probes failed to do thus far: the live‐cell imaging of MAO‐B activities without encountering diffusion problems.     

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.     

Boron Difluoride Curcuminoid Fluorophores with Enhanced Two‐Photon Excited Fluorescence Emission and Versatile Living‐Cell Imaging Properties

The synthesis of boron difluoride complexes of a series of curcuminoid derivatives containing various donor end groups is described. Time‐dependent (TD)‐DFT calculations confirm the charge‐transfer character of the second lowest‐energy transition band and ascribe the lowest energy band to a “cyanine‐like” transition. Photophysical studies reveal that tuning the donor strength of the end groups allows covering a broad spectral range, from the visible to the NIR region, of the UV–visible absorption and fluorescence spectra. Two‐photon‐excited fluorescence and Z‐scan techniques prove that an increase in the donor strength or in the rigidity of the backbone results in a considerable increase in the two‐photon cross section, reaching 5000 GM, with predominant two‐photon absorption from the S₀–S₂ charge‐transfer transition. Direct comparisons with the hemicurcuminoid derivatives show that the two‐photon active band for the curcuminoid derivatives has the same intramolecular charge‐transfer character and therefore arises from a dipolar structure. Overall, this structure–relationship study allows the optimization of the two‐photon brightness (i.e., 400–900 GM) with one dye that emits in the NIR region of the spectrum. In addition, these dyes demonstrate high intracellular uptake efficiency in Cos7 cells with emission in the visible region, which is further improved by using porous silica nanoparticles as dye vehicles for the imaging of two mammalian carcinoma cells type based on NIR fluorescence emission.