Bioluminescent Microcapsules: Applications in Activating a Photosensitizer

Bioluminescent microcapsules uploading D ‐luciferin have been fabricated by using the covalent assembly of firefly luciferase and alginate dialdehyde through a layer‐by‐layer technique. Such assembled microcapsules can produce visible light in the region of 520–680 nm, which can activate the photosensitizers rose bengal (RB) and hypocrellin B (HB) after adding ATP. The microcapsules uploading photosensitizers (RB or HB) have an obvious property to prevent the proliferation of tumor cells in the dark. The assembled bioluminescent microcapsules can be potentially used as photon donors for bioimaging, ATP detection, and photodynamic therapy.     

Giant Biodegradable Poly(ethylene glycol)‐block‐Poly(ε‐caprolactone) Polymersomes by Electroformation

Here, the formation of giant enzyme‐degradable polymersomes using the electroformation method is reported. Poly(ethylene glycol)‐block‐poly(ε‐caprolactone) polymersomes have been shown previously to be attractive candidates for the detection of bacterial proteases and protease mediated release of encapsulated reporter dyes and antimicrobials. To maximize the efficiency, the maximization of block copolymer (BCP) vesicle size without compromising their properties is of prime importance. Thus, the physical‐chemical properties of the BCP necessary to self‐assemble into polymeric vesicles by electroformation are first identified. Subsequently, the morphology of the self‐assembled structures is extensively characterized by different microscopy techniques. The vesicular structures are visualized for giant polymersomes by confocal laser scanning microscopy upon incorporation of reporter dyes during the self‐assembly process. Using time correlated single photon counting and by analyzing the fluorescence decay curves, the nanoenvironment of the encapsulated fluorophores is unveiled. Using this approach, the hollow core structure of the polymersomes is confirmed. Finally, the encapsulation of different dyes added during the electroformation process is studied. The results underline the potential of this approach for obtaining microcapsules for subsequent triggered release of signaling fluorophores or antimicrobially active cargo molecules that can be used for bacterial infection diagnostics and/or treatment.     

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.     

Two‐Photon Absorption and Time‐Resolved Stimulated Emission Depletion Spectroscopy of a New Fluorenyl Derivative

The synthesis, comprehensive linear photophysical characterization, two‐photon absorption (2PA), steady‐state and time‐resolved stimulated emission depletion properties of a new fluorene derivative, (E)‐1‐(2‐(di‐p‐tolylamino)‐9,9‐diethyl‐9H‐fluoren‐7‐yl)‐3‐(thiophen‐2‐yl)prop‐2‐en‐1‐one ( 1 ), are reported. The primary linear spectral properties, including excitation anisotropy, fluorescence lifetimes, and photostability, were investigated in a number of aprotic solvents at room temperature. The degenerate 2PA spectra of 1 were obtained with open‐aperture Z‐scan and two‐photon induced fluorescence methods, using a 1 kHz femtosecond laser system, and maximum 2PA cross‐sections of ～400–600 GM were obtained. The nature of the electronic absorption processes in 1 was investigated by DFT‐based quantum chemical methods implemented in the Gaussian 09 program. The one‐ and two‐photon stimulated emission spectra of 1 were measured over a broad spectral range using a femtosecond pump–probe‐based fluorescence quenching technique, while a new methodology for time‐resolved fluorescence emission spectroscopy is proposed. An effective application of 1 in fluorescence bioimaging was demonstrated by means of one‐ and two‐photon fluorescence microscopy images of HCT 116 cells containing dye encapsulated micelles.     

Bioconjugatable Azo‐Based Dark‐Quencher Dyes: Synthesis and Application to Protease‐Activatable Far‐Red Fluorescent Probes

We describe the efficient synthesis and one‐step derivatization of novel, nonfluorescent azo dyes based on the Black Hole Quencher‐3 (BHQ‐3) scaffold. These dyes were equipped with various reactive and/or bioconjugatable groups (azido, α‐iodoacetyl, ketone, terminal alkyne, vicinal diol). The azido derivative was found to be highly reactive in the context of copper‐catalyzed azide–alkyne cycloaddition (CuAAC) reactions and allowed easy synthetic access to the first water‐soluble (sulfonated derivative) and aldehyde‐modified BHQ‐3 dyes, the direct preparation of which failed by means of conventional azo‐coupling reactions. The aldehyde‐ and α‐iodoacetyl‐containing fluorescence quenchers were readily conjugated to aminooxy‐ and cysteine‐containing peptides by the formation of a stable oxime or thioether linkage, respectively. Further fluorescent labeling of the resultant peptide conjugates with red‐ or far‐red‐emitting rhodamine or cyanine dyes through sequential and/or one‐pot bioconjugations, led to novel Förster resonance energy transfer (FRET) based probes suitable for the in vivo detection and imaging of urokinase plasminogen activator, a key protease in cancer invasion and metastasis.     

Poly(1,4‐diketo‐3,6‐diphenylpyrrolo[3,4‐c]pyrrole‐alt−3,6‐carbazole/2,7‐fluorene) as high‐performance two‐photon dyes

This article reports the synthesis, one‐ and two‐photon absorption, and excited fluorescence properties of poly(1,4‐diketo‐3,6‐diphenylpyrrolo[3,4‐c]pyrrole‐alt‐N‐octyl‐3,6‐carbazole/2,7‐fluorene) ( PDCZ / PDFL ). PDCZ and PDFL are synthesized by the Suzuki cross‐coupling of 2,5‐dioctyl‐1,4‐diketo‐3,6‐bis(p‐bromophenylpyrrolo[3,4‐c]pyrrole and N‐octyl‐3,6‐bis(3,3‐dimethyl‐1,3,2‐dioxaborolan‐2‐yl)carbazole or 2,7‐bis(3,3‐dimethyl‐1,3,2‐dioxaborolan‐2‐yl)fluorene and have number‐average molecular weights of 8.5 × 10³ and 1.14 × 10⁴ g/mol and polydispersities of 2.06 and 1.83, respectively. They are highly soluble in common organic solvents and emit strong orange one‐ and two‐photon excited fluorescence (2PEF) in THF solution and exhibit high light and heat stability. The maximal two‐photon absorption cross‐sections (δ) measured in THF solution by the 2PEF method using femtosecond laser pulses are 970 and 900 GM per repeating unit for PDCZ and PDFL , respectively. These 1,4‐diketo‐pyrrolo[3,4‐c]pyrrole‐containing polymers with full aromatic structure and large δ will be promising high‐performance 2PA dyes applicable in two‐photon science and technology. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 944–951     

From Dark to Light to Fluorescence Resonance Energy Transfer (FRET): Polarity‐Sensitive Aggregation‐Induced Emission (AIE)‐Active Tetraphenylethene‐Fused BODIPY Dyes with a Very Large Pseudo‐Stokes Shift

The work presented herein is devoted to the fabrication of large Stokes shift dyes in both organic and aqueous media by combining dark resonance energy transfer (DRET) and fluorescence resonance energy transfer (FRET) in one donor–acceptor system. In this respect, a series of donor–acceptor architectures of 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) dyes substituted by one, two, or three tetraphenylethene (TPE) luminogens were designed and synthesised. The photophysical properties of these three chromophore systems were studied to provide insight into the nature of donor–acceptor interactions in both THF and aqueous media. Because the generation of emissive TPE donor(s) is strongly polarity dependent, due to its aggregation‐induced emission (AIE) feature, one might expect the formation of appreciable fluorescence emission intensity with a very large pseudo‐Stokes shift in aqueous media when considering FRET process. Interestingly, similar results were also recorded in THF for the chromophore systems, although the TPE fragment(s) of the dyes are non‐emissive. The explanation for this photophysical behaviour lies in the DRET. This is the first report on combining two energy‐transfer processes, namely, FRET and DRET, in one polarity‐sensitive donor–acceptor pair system. The accuracy of the dark‐emissive donor property of the TPE luminogen is also presented for the first time as a new feature for AIE phenomena.     

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.     

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.     

Alternating copolymers of 2,6‐bis(p‐dihexylaminostyryl)anthracene‐9,10‐diyl and N‐octylcarbazole with large two‐photon cross sections

We report the synthesis, thermal, one‐ and two‐photon properties of poly(2,6‐bis(p‐dihexylaminostyryl)anthracene‐9,10‐diyl‐alt‐N‐octylcarbazole‐3,6‐/2,7‐diyl) ( P1/P2 ). The as‐synthesized polymers exhibit number‐average molecular weights of 1.7 × 10⁴ for P1 and 2.1 × 10⁴ g/mol for P2 . They emit strong one‐ and two‐photon excitation fluorescence with the peak around 502 nm, and the fluorescence quantum yields around 0.76 in chloroform. In film state, P1 and P2 show different red‐shift emission with the peaks at 512 nm and 523 nm, respectively. The DSC measurement reveals that as‐synthesized polymers are all amorphous aggregates with the glass transition temperatures of 131 °C for P1 and 152 °C for P2 . The solution two‐photon absorption (TPA) properties of P1 and P2 in chloroform are measured by the two‐photon‐induced fluorescence method using femtosecond laser pulses (120 fs). The TPA cross sections (δ) are measured over the range of 700–900 nm. The maximal δ of P1 and P2 all appear at ～800 nm and are 1010 GM and 940 GM per repeating unit, respectively. This suggests that no notable interactions among structure units that impair their fluorescence and TPA properties, and the polymers with large δ can be obtained by using the high TPA‐active units as building blocks. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Synthesis and Photophysics of BF2‐Rigidified Partially Closed Chain Bromotetrapyrroles: Near Infrared Emitters and Photosensitizers

We report the synthesis, crystallographic, optical, and triplet and singlet oxygen generation properties of a series of BF₂‐rigidified partially closed chain bromotetrapyrroles as near infrared emitters and photosensitizers. These novel dyes were efficiently synthesized from a nucleophilic substitution reaction between pyrroles and the 3,5‐bromo‐substituents on the tetra‐ and hexabromoBODIPYs and absorb in the near‐infrared region (681–754 nm) with high molar extinction coefficients. Their fluorescent emission (708–818 nm) and singlet oxygen generation properties are significantly affected by alkyl substitutions on the two uncoordinated pyrrole units of these dyes and the polarity of solvents. Among them, dyes 4 ca and 4 da show good singlet oxygen generation efficiency and good NIR fluorescence emission (fluorescence quantum yields of 0.14–0.43 in different solvents studied).     

Two‐Photon‐Induced Fluorescence in New π‐Expanded Diketopyrrolopyrroles

Structurally unique π‐expanded diketopyrrolopyrroles (EDPP) were designed and synthesized. Strategic placement of a fluorene scaffold at the periphery of a diketopyrrolopyrrole through tandem Friedel–Crafts‐dehydration reactions resulted in dyes with supreme solubility. The structure of the dyes was confirmed by X‐ray crystallography verifying a nearly flattened arrangement of the ten fused rings. Despite the extended ring system, the dye still preserved good solubility and was further functionalized by using Pd‐catalyzed coupling reactions, such as the Buchwald–Hartwig amination. Photophysical studies of these new functional dyes revealed that they possess enhanced properties when compared with expanded DPPs in terms of two‐photon absorption cross‐section. It is further demonstrated that in addition to the initial diacetals, the final electrophilic cyclization step can also be applied to diketones. By placing two amine groups at peripheral positions of the resulting dyes, values of two‐photon absorption cross‐section on the level of 2000 GM around 1000 nm were achieved, which in combination with high fluorescence quantum yield (Φ_fl), generated a two‐photon brightness of approximately 1600 GM. These characteristics in combination with strong red emission (665 nm) make these new π‐expanded diketopyrrolopyrroles of major promise as two‐photon dyes for bioimaging applications. Finally, the corresponding N‐alkylated DPPs displayed a solid‐state fluorescence.     

Single Polyelectrolyte Microcapsules Fabricated By Glutaraldehyde‐Mediated Covalent Layer‐By‐Layer Assembly

Summary: Single polyelectrolyte component microcapsules and multilayers, exemplified by poly(allylamine hydrochloride) (PAH), have been prepared using a method of glutaraldehyde (GA)‐mediated covalent layer‐by‐layer (LbL) assembly. The GA cross‐linking of the adsorbed PAH results in surfaces covered by reactive aldehyde groups, which can then react with PAH to result in another layer of covalently linked PAH. The repeated assembly of single polyelectrolyte in an LbL manner can be thus achieved. The PAH multilayers can grow linearly along with the layer number, and their thickness can be controlled at the nanometer scale, as verified by UV‐vis absorption spectrometry and ellipsometry. Single polyelectrolyte microcapsules are obtained after removal of the template cores at low pH. The morphology and integrity are confirmed by scanning force microscopy and confocal laser scanning microscopy.

Schematic illustration of the preparation of a single polyelectrolyte component microcapsule by GA‐mediated covalent LbL assembly.     

Light‐Harvesting Nanoparticle Probes for FRET‐Based Detection of Oligonucleotides with Single‐Molecule Sensitivity

Controlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20‐nm light‐harvesting nanoantenna particles, built of a tailor‐made hydrophobic charged polymer poly(ethyl methacrylate‐co‐methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87‐fold brighter than quantum dots QDots 605 in single‐particle microscopy (with 550‐nm excitation), these DNA‐functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250‐fold signal amplification. The obtained FRET nanoprobes enable single‐molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi‐fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight.     

A Small‐Molecule FRET Reporter for the Real‐Time Visualization of Cell‐Surface Proteolytic Enzyme Functions

Real‐time imaging of cell‐surface‐associated proteolytic enzymes is critical to better understand their performances in both physiological and pathological processes. However, most current approaches are limited by their complexity and poor membrane‐anchoring properties. Herein, we have designed and synthesized a unique small‐molecule fluorescent probe, which combines the principles of passive exogenous membrane insertion and Förster resonance energy transfer (FRET) to image cell‐surface‐localized furin‐like convertase activities. The membrane‐associated furin‐like enzymatic cleavage of the peptide probe leads to an increased fluorescence intensity which was mainly localized on the plasma membrane of the furin‐expressed cells. This small‐molecule fluorescent probe may serve as a unique and reliable reporter for real‐time visualization of endogenous cell‐surfaceassociated proteolytic furin‐like enzyme functions in live cells and tissues using one‐photon and two‐photon microscopy.     

Precise Labeling and Tracking of Lipid Droplets in Adipocytes Using a Luminescent ZnSalen Complex

Designing two‐photon probes for precise labeling of lipid droplets (LDs) and monitoring LD dynamics in adipocytes is of great significance to understand LD homeostasis. We herein report that a luminescent metal complex LD‐TPZn can specifically image LDs in adipose cells and tissue using one‐ or two‐photon fluorescence microscopy. Importantly, LD‐TPZn exhibited higher specificity to LDs than the commercial dyes Nile Red and Bodipy 493/503, probably due to different cellular uptake pathways, that is, clathrin‐mediated endocytosis and non‐selectively passive diffusion, respectively. More importantly, LD‐TPZn can be applied as a two‐photon LD probe to image adipose tissue, one of the most challengeable tissues for traditional one‐photon fluorescence microscope imaging due to the strong light scattering. Most importantly, LD‐TPZn can be used to monitor LD growth during adipogenesis of preadipocytes, which is highly desirable to unravel the relationship between LD homeostasis and metabolic diseases.     

Theoretical and Experimental Study on Boron β‐Diketonate Complexes with Intense Two‐Photon‐Induced Fluorescence in Solution and in the Solid State

Three boron diketonate chromophores with extended π‐conjugated backbone were prepared and their spectroscopic features were investigated through a combined theoretical/experimental study. It was shown that these complexes, which undergo very large electronic reorganization upon photoexcitation, combine large two‐photon absorption cross section with an emission energy and quantum efficiency in solution that is strongly dependent on solvent polarity. The strong positive influence of boron complexation on the magnitude of the two‐photon absorption was clearly established, and it was shown that the two‐photon absorption properties were dominated by the quadrupolar term. For one of the synthesized compounds, intense one‐ and two‐photon‐induced solid‐state emission (fluorescence quantum yield of 0.65 with maximum wavelength of 610 nm) was obtained as a result of antiparallel J‐aggregate crystal packing.     

Rational Design of Fluorescent Phthalazinone Derivatives for One‐ and Two‐Photon Imaging

Phthalazinone derivatives were designed as optical probes for one‐ and two‐photon fluorescence microscopy imaging. The design strategy involves stepwise extension and modification of pyridazinone by 1) expansion of pyridazinone to phthalazinone, a larger conjugated system, as the electron acceptor, 2) coupling of electron‐donating aromatic groups such as N,N‐diethylaminophenyl, thienyl, naphthyl, and quinolyl to the phthalazinone, and 3) anchoring of an alkyl chain to the phthalazinone with various terminal substituents such as triphenylphosphonio, morpholino, triethylammonio, N‐methylimidazolio, pyrrolidino, and piperidino. Theoretical calculations were utilized to verify the initial design. The desired fluorescent probes were synthesized by two different routes in considerable yields. Twenty‐two phthalazinone derivatives were synthesized and their photophysical properties were measured. Selected compounds were applied in cell imaging, and valuable information was obtained. Furthermore, the designed compounds showed excellent performance in two‐photon microscopic imaging of mouse brain slices.     

Synthesis,one‐ and two‐photon properties of poly[9,10‐bis(3,4‐bis(2‐ethylhexyl‐oxy)phenyl)‐2,6‐anthracenevinylene‐alt‐N‐octyl‐3,6‐/2,7‐carbazolevinylene]

The synthesis, one‐ and two‐photon absorption (TPA) and emission properties of two novel 2,6‐anthracenevinylene‐based copolymers, poly[9,10‐bis(3,4‐bis(2‐ethylhexyloxy)phenyl)‐2,6‐anthracenevinylene‐alt‐N‐octyl‐3,6‐carbazolevinyl‐ene] ( P1 ) and poly[9,10‐bis(3,4‐bis(2‐ethylhexyloxy)phenyl)‐2,6‐anthracenevinyl‐ene‐alt‐N‐octyl‐2,7‐carbazolevinylene] ( P2 ) were reported. The as‐synthesized polymers have the number‐average molecular weights of 1.56 × 10⁴ for P1 and 1.85 × 10⁴ g mol^?1 for P2 and are readily soluble in common organic solvents. They emit strong bluish‐green one‐ and two‐photon excitation fluorescence in dilute toluene solution (? _P1 = 0.85, ? _P2 = 0.78, λ_em( P1 ) = 491 nm, λ_em( P2 ) = 483 nm). The maximal TPA cross‐sections of P1 and P2 measured by the two‐photon‐induced fluorescence method using femtosecond laser pulses in toluene are 840 and 490 GM per repeating unit, respectively, which are obviously larger than that (210 GM) of poly[9,10‐bis‐(3,4‐bis(2‐ethylhexyloxy) phenyl)‐2,6‐anthracenevinylene], indicating that the poly(2,6‐anthracenevinylene) derivatives with large TPA cross‐sections can be obtained by inserting electron‐donating moieties into the polymer backbone. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 463–470, 2010     

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.