Development of Photoactivated Fluorescent N‐Hydroxyoxindoles and Their Application for Cell‐Selective Imaging

Photoactivatable fluorophores are essential tools for studying the dynamic molecular interactions within important biological systems with high spatiotemporal resolution. However, currently developed photoactivatable fluorophores based on conventional dyes have several limitations including reduced photoactivation efficiency, cytotoxicity, large molecular size, and complicated organic synthesis. To overcome these challenges, we herein report a class of photoactivatable fluorescent N‐hydroxyoxindoles formed through the intramolecular photocyclization of substituted o‐nitrophenyl ethanol (ONPE). These oxindole fluorophores afford excellent photoactivation efficiency with ultra‐high fluorescence enhancement (up to 800‐fold) and are small in size. Furthermore, the oxindole derivatives show exceptional biocompatibility by generating water as the only photolytic side product. Moreover, structure–activity relationship analysis clearly revealed the strong correlation between the fluorescent properties and the substituent groups, which can serve as a guideline for the further development of ONPE‐based fluorescent probes with desired photophysical and biological properties. As a proof‐of‐concept, we demonstrated the capability of a new substituted ONPE that has an uncaging wavelength of 365–405 nm and an excitation/emission at 515 and 620 nm, for the selective imaging of a cancer cell line (Hela cells) and a human neural stem cell line (hNSCs).     

A Pyrene Derivative for Hg2+‐Selective Fluorescent Sensing and Its Application in In Vivo Imaging

An Hg²⁺‐selective fluorescent sensor ( 1 ) bearing pyrene as a fluorophore was synthesized. A sandwich‐stacking binding mode was formed during the binding process, which increased the excimer fluorescence 22‐fold at 490 nm. Compound 1 was successfully applied in in vivo imaging to trace the enrichment and distribution of mercury in the nervous system, digestive system, and reproductive system of Caenorhabditis elegans, as well as the organs of zebrafish.     

Red‐Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy

Fluorescent markers emitting in the red are extremely valuable in biological microscopy since they minimize cellular autofluorescence and increase flexibility in multicolor experiments. Novel rhodamine dyes excitable with 630 nm laser light and emitting at around 660 nm have been developed. The new rhodamines are very photostable and have high fluorescence quantum yields of up to 80 %, long excited state lifetimes of 3.4 ns, and comparatively low intersystem‐crossing rates. They perform very well both in conventional and in subdiffraction‐resolution microscopy such as STED (stimulated emission depletion) and GSDIM (ground‐state depletion with individual molecular return), as well as in single‐molecule‐based experiments such as fluorescence correlation spectroscopy (FCS). Syntheses of lipophilic and hydrophilic derivatives starting from the same chromophore‐containing scaffold are described. Introduction of two sulfo groups provides high solubility in water and a considerable rise in fluorescence quantum yield. The attachment of amino or thiol reactive groups allows the dyes to be used as fluorescent markers in biology. Dyes deuterated at certain positions have narrow and symmetrical molecular mass distribution patterns, and are proposed as new tags in MS or LC‐MS for identification and quantification of various substance classes (e.g., amines and thiols) in complex mixtures. High‐resolution GSDIM images and live‐cell STED‐FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy.     

A New Highly Selective and Sensitive Assay for Fluorescence Imaging of .OH in Living Cells: Effectively Avoiding the Interference of Peroxynitrite

A new nonredox fluorescent probe to realize the imaging of hydroxyl radicals (^.OH) in living cells was designed and synthesized. The structure comprised the fluorescent dye boron dipyrromethene (BDP) and a 2,2,6,6‐tetramethyl‐1‐piperidinoxyl (TEMPO) unit. This probe could rapidly respond to ^.OH with a detection limit of 18 pM , and it possessed superior photostability and pH insensitivity. Other reactive oxygen species (ROS) and relevant intracellular components did not interfere. In particular, the important problem of ONOO^? interference was efficiently avoided. An MTT assay proved that the probe was not very cytotoxic. The probe could penetrate into intact cell membranes to selectively detect intracellular ^.OH without causing cellular damage in living mice macrophages, normal human liver cells. and human hepatoma cells. These advantageous characteristics make the fluorescent probe potentially useful as a new candidate to detect ^.OH in broad biosystems.     

Nanobody‐Conjugated Nanotubes for Targeted Near‐Infrared In Vivo Imaging and Sensing

Fluorescent nanomaterials such as single‐walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP‐binding nanobodies to DNA‐wrapped SWCNTs. This approach combines the targeting capabilities of GFP‐binding nanobodies and the nonbleaching near‐infrared fluorescence (850–1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin‐5‐GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.     

Fluorescence Nanoscopy with Optical Sectioning by Two‐Photon Induced Molecular Switching using Continuous‐Wave Lasers

During the last decade far‐field fluorescence microscopy methods have evolved that have resolution far below the wavelength of light. To outperform the limiting role of diffraction, all these methods, in one way or another, switch the ability of a molecule to emit fluorescence. Here we present a novel rhodamine amide that can be photoswitched from a nonfluorescent to a fluorescent state by absorption of one or two photons from a continuous‐wave laser beam. This bright marker enables strict control of on/off switching and provides single‐molecule localization precision down to 15 nm in the focal plane. Two‐photon induced nonlinear photoswitching of this marker with continuous‐wave illumination offers optical sectioning with simple laser equipment. Future synthesis of similar compounds holds great promise for cost‐effective fluorescence nanoscopy with noninvasive optical sectioning.     

Sterically Shielded Heptamethine Cyanine Dyes for Bioconjugation and High Performance Near‐Infrared Fluorescence Imaging

The near‐infrared window of fluorescent heptamethine cyanine dyes greatly facilitates biological imaging because there is deep penetration of the light and negligible background fluorescence. However, dye instability, aggregation, and poor pharmacokinetics are current drawbacks that limit performance and the scope of possible applications. All these limitations are simultaneously overcome with a new molecular design strategy that produces a charge balanced and sterically shielded fluorochrome. The key design feature is a meso‐aryl group that simultaneously projects two shielding arms directly over each face of a linear heptamethine polyene. Cell and mouse imaging experiments compared a shielded heptamethine cyanine dye (and several peptide and antibody bioconjugates) to benchmark heptamethine dyes and found that the shielded systems possess an unsurpassed combination of photophysical, physiochemical, and biodistribution properties that greatly enhance bioimaging performance.     

Benzo[1,2‐d:4,5‐d′]bisimidazoles as a Convenient Platform Towards Dyes that are Capable of Excited‐State Intramolecular Proton Transfer and of Two‐Photon Absorption

New, strongly fluorescent benzo[1,2‐d:4,5‐d′]bisimidazoles have been prepared by the reaction of Bandrowski′s base with various aldehydes. Their structures were carefully designed to achieve efficient excited‐state intramolecular proton transfer and good two‐photon‐absorption (2PA) cross‐sections. Functional dyes that possessed both high fluorescence quantum yields and large Stokes shifts were prepared. A π‐expanded D‐A‐D derivative that possessed Φ_fl=50 % and σ₂=230 GM in the spectroscopic area of interest for biological imaging is an excellent candidate as a fluorescent probe. Thanks to the presence of two reactive amino groups, such compounds can be easily transformed into probes for bioconjugation. All of these benzo[1,2‐d:4,5‐d′]bisimidazoles were also strongly fluorescent in the solid state.     

Color-Tunable Light-up Bioorthogonal Probes for In Vivo Two-Photon Fluorescence Imaging

Light-up bioorthogonal probes have attracted increasing attention recently due to their capability to directly image diverse biomolecules in living cells without washing steps. The development of bioorthogonal probes with excellent fluorescent properties suitable for in vivo imaging, such as long excitation/emission wavelength, high fluorescence turn-on ratio, and deep penetration, has been rarely reported. Herein, a series of azide-based light-up bioorthogonal probes with tunable colors based on a weak fluorescent 8-aminoquinoline ( AQ ) scaffold were designed and synthesized. The azido quinoline derivatives are able to induce large fluorescence enhancement (up to 1352-fold) after click reaction with alkynes. In addition, the probes could be engineered to exhibit excellent two-photon properties (δ=542 GM at 780 nm) after further introducing different styryl groups into the AQ scaffold. Subsequent detailed bioimaging experiments demonstrated that these versatile probes can be successfully used for live cell/zebrafish imaging without washing steps. Further in vivo two-photon imaging experiments demonstrated that these light-up biorthogonal probe outperformed conventional fluorophores, for example, high signal-to-noise ratio and deep tissue penetration. The design strategy reported in this study is a useful approach to realize diverse high-performance biorthogonal light-up probes for in vivo studying.     

A Pyrene‐functionalized Polynorbornene for Ratiometric Fluorescence Sensing of Pyrophosphate

A novel pyrene‐functionalized polynorbornene ( P1 ) bearing sulfonamide NH and triazolium donors has been synthesized for ratiometric fluorescence sensing of PPi in aqueous solution. In addition, P1 is also used to monitor intracellular PPi and to detect PPi released during polymerase chain reaction.     

A Selective Near‐Infrared Fluorescent Probe for In Vivo Imaging of Thiophenols from a Focused Library

Thiophenols are highly toxic industrial materials that, once released, will accumulate in the environment, and ultimately in human bodies, thereby causing serious health problems. To achieve their selective and sensitive detection, a novel near‐infrared (NIR) fluorescent probe ( CCP‐1 ) from a focused library was developed for thiophenol species. Our studies show that CCP‐1 displays a thiophenol‐triggered 28‐fold fluorescence intensity enhancement at 706 nm, with a detection limit of 34 nm observed. It is also able to differentiate thiophenols from various other thiol‐containing analytes including hydrogen sulfide, hydrogen persulfide, and aliphatic thiols. In total, the desirable properties (e.g., excitation/emission in the NIR region, good cell‐membrane permeability, intracellular stability, and low cytotoxicity) make CCP‐1 a potential candidate for thiophenol detection both in vitro and in vivo. In addition, CCP‐1 , for the first time, successfully visualized thiophenols in mice models of thiophenol inhalation.     

Photophysics of New Water‐Soluble Terrylenediimide Derivatives and Applications in Biology

The photophysical properties of three new water‐soluble terrylenediimide (WS‐TDI) derivatives are investigated and their utilization in biological experiments is demonstrated. Each of these dyes can be excited in the far red region of the visible spectrum, making them good candidates for in‐vivo studies. Single‐molecule techniques characterize their photophysics that is, the number of emitted photons, blinking characteristics and survival times until photobleaching takes place. All three dyes exhibit bright fluorescence, as well as an extremely high resistance against photodegradation compared to other well‐known fluorophores. Due to their different characteristics the three new WS‐TDI derivatives are suitable for specialized biological applications. WS‐TDI dodecyl forms non‐fluorescent aggregates in water which can be disrupted in a hydrophobic environment leading to a monomeric fluorescent form. Due to its high lipophilicity WS‐TDI dodecyl anchors efficiently in lipid bilayers with its alkyl chain and hence can be ideally used to image membranes and membrane‐containing compartments in living cells. In contrast, the positively charged WS‐TDI pyridoxy is a new type of chromophore in the WS‐TDI family. It is fully solubilized in water forming fluorescent monomers and is successfully used to label the envelope of herpes simplex viruses. Finally, it is shown that a WS‐TDI derivative functionalized with N‐hydroxysuccinimide ester moiety (WS‐TDI/NHS ester) provides a versatile reactive dye molecule for the specific labelling of amino groups in biomolecules such as DNA.     

A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)‐Enhanced Photostability and a Narrow Band of Emission

We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)‐sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET‐conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)‐enhanced emission of 3 and 4 in lysosomes. It was further shown using two‐color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino‐functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.     

Sterically Shielded Heptamethine Cyanine Dyes for Bioconjugation and High Performance Near-Infrared Fluorescence Imaging

The near-infrared window of fluorescent heptamethine cyanine dyes greatly facilitates biological imaging because there is deep penetration of the light and negligible background fluorescence. However, dye instability, aggregation, and poor pharmacokinetics are current drawbacks that limit performance and the scope of possible applications. All these limitations are simultaneously overcome with a new molecular design strategy that produces a charge balanced and sterically shielded fluorochrome. The key design feature is a meso-aryl group that simultaneously projects two shielding arms directly over each face of a linear heptamethine polyene. Cell and mouse imaging experiments compared a shielded heptamethine cyanine dye (and several peptide and antibody bioconjugates) to benchmark heptamethine dyes and found that the shielded systems possess an unsurpassed combination of photophysical, physiochemical, and biodistribution properties that greatly enhance bioimaging performance.     

Modified Isoindolediones as Bright Fluorescent Probes for Cell and Tissue Imaging

New L -shaped fluorophores possessing five conjugated rings have been synthesized through a four-step procedure involving diketopyrrolopyrrole synthesis and its double N-alkylation, followed by trimethylsilyl bromide-mediated rearrangement to thieno[2,3-f]isoindole-5,8-dione and an intramolecular Friedel–Crafts reaction. In comparison with the parent isoindolediones and π-expanded diketopyrrolopyrroles, these new dyes show red-shifted absorption and emission (up to ≈630 nm). Their structural rigidity is responsible for both the observed small Stokes shifts and large fluorescence quantum yields. Tissue imaging studies revealed that these new dyes show advantageous features including minimal autofluorescence interference and pronounced solvent-sensitive emission. Interestingly, there is a fundamental difference between a dye possessing an amino group and its analog bearing an N-alkyl substituent. The former dye under two-photon excitation at 900 nm gives bright images whereas its N-alkylated counterpart does not. A new type of membrane localization has been discovered by an N-alkylated isoindoledione possessing a benzofuryl substituent. In spite of the fact that the fluorescence quantum yield of this dye in a range of solvents is rather low, it does stain cell membranes exclusively. This new mode of cellular staining opens the door towards further development of membrane staining dyes.     

Fluoride Sensing by Catechol‐Based π‐Electron Systems

We have developed new catechol‐based sensors that can detect fluoride via fluorescence or optical absorption even in the presence of other halides. The level and sensitivity of detection of the sensing molecules is dependent on the chromophore length, which is controlled by the number of thiophene units (one to three) within the chromophore. The sensor with three thiophene units, (E)‐2‐(2,2′‐terthiophen‐5‐yl)‐3‐(3,4‐dihydroxyphenyl)acrylonitrile, gives the best response to fluoride. By using fluorescence measurements fluoride is detectable over the concentration range 1.7 μM to 200 μM . Importantly, when adsorbed onto a solid support the fluorescent catechol dye can be used to detect the presence of fluoride in aqueous solution.     

Polar Diketopyrrolopyrrole‐Imidazolium Salts as Selective Probes for Staining Mitochondria in Two‐Photon Fluorescence Microscopy

Three rationally designed polar derivatives of diketopyrrolopyrrole consisting of 1,3‐dimethylimidazolium cationic units and benzene, thiophene, or furan rings as π spacers were synthesized and thoroughly studied. The obtained salts are soluble in polar organic solvents and show satisfactory solubility in water, which makes them suitable for the applications in bioimaging. Photophysical measurements revealed that the obtained derivatives are characterized by strong absorption and good fluorescence quantum yields. The corresponding two‐photon properties were also examined and showed that the synthesized salts exhibit large two‐photon absorption cross‐sections reaching 4000 GM (GM=Goeppert‐Mayer unit, 1 GM=10^?50 cm⁴ s photon^?1) and very high two‐photon brightness values exceeding 2000 GM. It was demonstrated that these salts can be safely applied in two‐photon fluorescence microscopy for selective staining of mitochondria in living cells.