Red‐ and Green‐Emitting Iridium(III) Complexes for a Dual Barometric and Temperature‐Sensitive Paint

A new dual luminescent sensitive paint for barometric pressure and temperature (T) is presented. The green‐emitting iridium(III) complex [Ir(ppy)₂(carbac)] (ppy=2‐phenylpyridine; carbac=1‐(9H‐carbazol‐9‐yl)‐5,5‐dimethylhexane‐2,4‐dione) was applied as a novel probe for T along with the red‐emitting complex [Ir(btpy)₃], (btpy=2‐(benzo[b]thiophene‐2‐yl)pyridine) which functions as a barometric (in fact oxygen‐sensitive) probe. Both iridium complexes were dissolved in different polymer materials to achieve optimal responses. The probe [Ir(ppy)₂(carbac)] was dispersed in gas‐blocking poly(acrylonitrile) microparticles in order to suppress any quenching of its luminescence by oxygen. The barometric probe [Ir(btpy)₃], in turn, was incorporated in a cellulose acetate butyrate film which exhibits good permeability for oxygen. The effects of temperature on the response of the oxygen probe can be corrected by simultaneous optical determination of T, as the poly(acrylonitrile) microparticles containing the temperature indicator are incorporated into the film. The phosphorescent signals of the probes for T and barometric pressure, respectively, can be separated by optical filters due to the ≈75 nm difference in their emission maxima. The dual sensor is applicable to luminescence lifetime imaging of T and barometric pressure. It is the first luminescent dual sensor material for barometric pressure/T based exclusively on the use of Ir^III complexes in combination with luminescence lifetime imaging.     

Highly Fluorescent and Water‐Soluble Diketopyrrolopyrrole Dyes for Bioconjugation

The preparation of highly water‐soluble and strongly fluorescent diketopyrrolopyrrole (DPP) dyes using an unusual taurine‐like sulfonated linker has been achieved. Exchanging a phenyl for a thienyl substituent shifts the emission wavelength to near λ=600 nm. The free carboxylic acid group present in these new derivatives was readily activated and the dyes were subsequently covalently linked to a model protein (bovine serum albumin; BSA). The bioconjugates were characterized by electronic absorption, fluorescence spectroscopy and MALDI‐TOF mass spectrometry, thus enabling precise determination of the labeling density (ratio DPP/BSA about 3 to 8). Outstanding values of fluorescence quantum yield (30 % to 59 %) for these bioconjugates are obtained. The photostability of these DPP dyes is considerably greater than that of fluorescein under the same irradiation conditions. Remarkably low detection limits between 80 and 300 molecules/μm² were found for the BSA bioconjugates by fluorescence imaging with a epifluorescence microscope.     

A Cell‐Targeted Non‐Cytotoxic Fluorescent Nanogel Thermometer Created with an Imidazolium‐Containing Cationic Radical Initiator

A cationic fluorescent nanogel thermometer based on thermo‐responsive N‐isopropylacrylamide and environment‐sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02–0.84 °C in the range 20–40 °C), and remarkable non‐cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells.     

Bifunctional Fluorescent Probe for Sequential Sensing of Thiols and Primary Aliphatic Amines in Distinct Fluorescence Channels

Thiols and primary aliphatic amines (PAA) are ubiquitous and extremely important species in biological systems. They perform significant interplaying roles in complex biological events. A single fluorescent probe differentiating both thiols and PAA can contribute to understanding the intrinsic inter‐relationship of thiols and PAA in biological processes. Herein, we rationally constructed the first fluorescent probe that can respond to thiols and PAA in different fluorescence channels. The probe exhibited a high selectivity and sensitivity to thiols and PAA. In addition, it displayed sequential sensing ability when the thiols and PAA coexisted. The application experiments indicated that the probe can be used for sensing thiols and PAA in human blood serum. Moreover, the fluorescence imaging of endogenous thiols and PAA as well as antihypertensive drugs captopril and amlodipine in living cells were successfully conducted.     

Keto‐benzo[h]‐Coumarin‐Based Near‐Infrared Dyes with Large Stokes Shifts for Bioimaging Applications

Fluorescence imaging is a promising tool for the visualization of biomolecules in living systems and there is great demand for new fluorescent dyes that absorb and emit in the near‐infrared (NIR) region. Herein, we constructed three new fluorescent dyes ( NBC dyes) based on keto‐benzo[h]coumarin ( k‐BC ) and benzopyrilium salts. These dyes showed large Stokes shifts (>100 nm) and NIR emission (>800 nm). The relationship between the structures and optical properties of these dyes was further investigated by using density functional theory calculations at the B3LYP/6‐3G level of theory. Fluorescence images indicated that the fabricated dyes exhibited good photostability and low cytotoxicity and, thus, have potential applications as imaging agents in living cells and animals.     

Monofluorination and Trifluoromethylation of BODIPY Dyes for Prolonged Single‐Molecule Detection

Electrophilic monofluorination with Selectfluor and nucleophilic trifluoromethylation with the Ruppert–Prakesh reagent of dimethyl‐, tetramethyl‐ and pentamethyl‐substituted boron dipyrromethenes (BODIPY) are investigated. Monofluorinated dyes are synthesized with low yields (<30 %), however trifluoromethyl derivatives are obtained in moderate to high yields (≈40–90 %). All compounds are characterized by steady‐state and time‐resolved fluorescence spectroscopy, the photostability is investigated with fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRF). Monofluorination hardly affects the spectroscopic parameters of the unsubstituted parent compounds, but distinctly enhances the photostability, whereas trifluoromethylation leads to a hypsochromic shift by up to 17 nm in both absorption and emission, slightly enhanced intersystem crossing, and higher photostability. Further development of soft fluorination and trifluoromethylation methods is therefore highly desired.     

Iron(III)‐Selective Chelation‐Enhanced Fluorescence Sensing for In Vivo Imaging Applications

A “turn‐on” pattern Fe³⁺‐selective fluorescent sensor was synthesized and characterized that showed high fluorescence discrimination of Fe³⁺ over Fe²⁺ and other tested ions. With a 62‐fold fluorescence enhancement towards Fe³⁺, the probe was employed to detect Fe³⁺ in vivo in HeLa cells and Caenorhabditis elegans, and it was also successfully used to elucidate Fe³⁺ enrichment and exchange infected by innexin3 (Inx3) in hemichannel‐closed Sf9 cells.     

Reversible Chemical Reactions for Single‐Color Multiplexing Microscopy

Recent developments in biology demand an increasing number of simultaneously imaged structures with standard fluorescence microscopy. However, the number of multiplexed channels is limited for most multiplexing modalities, such as spectral multiplexing or fluorescence‐lifetime imaging. We propose extending the number of imaging channels by using chemical reactions, controlling the emissive state of fluorescent dyes. As proof of concept, we reversibly switch a fluorescent copper sensor to enable successive imaging of two different structures in the same spectral channel. We also show that this chemical multiplexing is orthogonal to existing methods. By using two different dyes, we combine chemical with spectral multiplexing for the simultaneous imaging of four different structures with only two spectrally different channels. We characterize and discuss the approach and provide perspectives for extending imaging modalities in stimulated emission depletion microscopy, for which spectral multiplexing is technically demanding.     

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.     

A Lysosome‐Compatible Near‐Infrared Fluorescent Probe for Targeted Monitoring of Nitric Oxide

The requirement for nitric oxide (NO) of lysosomes has motivated the development of a sophisticated fluorescent probe to monitor the distribution of this important biomolecule at the subcellular level in living cells. A near‐infrared (NIR) fluorescent Si‐rhodamine (SiRB)‐NO probe was designed based on the NO‐induced ring‐opening process of Si‐rhodamine. The probe exhibits fast chromogenic and fluorogenic responses, and high sensitivity and selectivity toward trace amounts of NO. Significantly, the spirolactam in Si‐rhodamine exhibits very good tolerance to H⁺, which in turn brings extremely low background fluorescence not only in the physiological environment but also under acidic conditions. The stability of the highly fluorescent product in acidic solution provides persistent fluorescence emission for long‐term imaging experiments. To achieve targeted imaging with improved spatial resolution and sensitivity, an efficient lysosome‐targeting moiety was conjugated to a SiRB‐NO probe, affording a tailored lysosome‐targeting NIR fluorescent Lyso‐SiRB‐NO probe. Inheriting the key advantages of its parent SiRB‐NO probe, Lyso‐SiRB‐NO is a functional probe that is suited for monitoring lysosomal NO with excellent lysosome compatibility. Imaging experiments demonstrated the monitoring of both exogenous and endogenous NO in real time by using the Lyso‐SiRB‐NO probe.     

New Red‐Emitting Tetrazine‐Phenoxazine Fluorogenic Labels for Live‐Cell Intracellular Bioorthogonal Labeling Schemes

The synthesis of a set of tetrazine‐bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through‐bond energy‐transfer‐based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse‐electron‐demand Diels–Alder reaction with proteins modified genetically with strained trans‐cyclooctenes.     

Synthesis and Spectroscopic Properties of Fused‐Ring‐Expanded Aza‐Boradiazaindacenes

A series of fused‐ring‐expanded aza‐boradiazaindacene (aza‐BODIPY) dyes have been synthesized by reacting arylmagnesium bromides with phthalonitriles or naphthalenedicarbonitriles. An analysis of the structure–property relationships has been carried out based on X‐ray crystallography, optical spectroscopy, and theoretical calculations. Benzo and 1,2‐naphtho‐fused 3,5‐diaryl aza‐BODIPY dyes display markedly red shifted absorption and emission bands in the near‐IR region (>700 nm) due to changes in the energies of the frontier MOs relative to those of 1,3,5,7‐tetraaryl aza‐BODIPYs. Only one 1,2‐naphtho‐fused aza‐BODIPY of the three possible isomers is formed due to steric effects, and 2,3‐naphtho‐fused compounds could not be characterized because the final BF₂ complexes are unstable in solution. The incorporation of a  N(CH₃)₂ group at the para‐positions of a benzo‐fused 3,5‐diaryl aza‐BODIPY quenches the fluorescence in polar solvents and results in a ratiometric pH response, which could be used in future practical applications as an NIR “turn‐on” fluorescence sensor.     

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).     

Nondestructive Photoluminescence Read‐Out by Intramolecular Electron Transfer in a Perylene Bisimide‐Diarylethene Dyad

A novel, highly stable photochromic dyad 3 based on a perylene bisimide (PBI) fluorophore and a diarylethene (DAE) photochrome was synthesized and the optical and photophysical properties of this dyad were studied in detail by steady‐state and time‐resolved ultrafast spectroscopy. This photochromic dyad can be switched reversibly by UV‐light irradiation of its ring‐open form 3 o leading to the ring‐closed form 3 c , and back reaction of 3 c to 3 o by irradiation with visible light. Solvent‐dependent fluorescence studies revealed that the emission of ring‐closed form 3 c is drastically quenched in solvents of medium (e.g., chloroform) to high (e.g., acetone) polarities, while the emission of the ring‐open form 3 o is appreciably quenched only in highly polar solvents like DMF. The strong fluorescence quenching of 3 c is attributed to a photoinduced electron‐transfer (PET) process from the excited PBI unit to ring‐closed DAE moiety, as this process is thermodynamically highly favorable with a Gibbs free energy value of ?0.34 eV in dichloromethane. The electron‐transfer mechanism for the fluorescence quenching of ring‐closed 3 c is substantiated by ultrafast transient measurements in dichloromethane and acetone, revealing stabilization of charge‐separated states of 3 c in these solvents. Our results reported here show that the new photochromic dyad 3 has potential for nondestructive read‐out in write/read/erase fluorescent memory systems.     

Two‐Photon Absorption Cross‐Sections of Reference Dyes: A Critical Examination

The electronic structure and one‐ and two‐photon absorption spectra of four fluorophores, p‐bis(o‐methoxystyryl)benzene (Bis‐MSB), coumarin 307, fluorescein and rhodamine B, commonly used as reference compounds for two‐photon absorption spectra, have been theoretically calculated and compared with available experimental data. The possible reasons for the wide discrepancies in two‐photon absorption cross‐sections reported in the literature are discussed on the basis of the theoretical findings. The role of a solvent environment on the electronic one‐ and two‐photon absorption spectra is also studied. We highlight some necessary precautions that one needs to take when comparing literature results of two‐photon absorption cross‐sections.     

Synthesis and Optical Properties of Water‐Soluble Polyglycerol‐Dendronized Rylene Bisimide Dyes

Four new water‐soluble polyglycerol‐dendronized perylene, terrylene, and quaterrylene bisimides have been synthesized and characterized with respect to their optical properties in polar organic solvents and water by using UV/Vis and fluorescence spectroscopy. All of these dyes were highly soluble in water, but the size of the chosen polyglycerol dendron was only sufficient to completely suppress dye aggregation for the core‐unsubstituted perylene derivative. Their high solubility in water and their absorption and emission wavelengths up to the NIR region make the core‐unsubstituted perylene and terrylene bisimides ideal candidates for applications in bioimaging, whilst the lack of fluorescence for quaterrylene bisimide in all polar solvents does not warrant further investigation of this chromophore in fluorescence and imaging applications. Likewise, tuning of the emission of rylene bisimides towards longer wavelengths by employing electron‐donating bay substituents is not a promising strategy, owing to the lower fluorescence quantum yields in polar solvents and, in particular, in water.     

Solid‐State Gas Sensors Developed from Functional Difluoroboradiazaindacene Dyes

This article describes the synthesis and characterization of several new difluoroboradiazaindacene (BODIPY?) dyes functionalized at the central 8‐position by a phenyliodo, phenylheptynoate or phenylheptynoic fragment and at the 3‐ or 3/5‐position(s) by 4‐dimethylaminophenylstyryl residue(s). Single‐crystal structural determinations confirm the planarity of the dyes, while the absorption and fluorescence spectroscopic properties are highly sensitive to the state of protonation (or alkylation) of the terminal anilino donor group(s). Reversible color tuning from green to blue for absorption and from colorless (i.e., near‐IR region) to red for fluorescence is obtained on successive addition of acid and base. The difunctionalized derivative is especially interesting in this respect and shows two well‐resolved pK_a values of 5.10 and 3.04 in acetonitrile. Addition of the first proton causes only small spectral changes and deactivates the molecule towards addition of the second proton. It is this latter step that accommodates the large change in absorption and emission properties, due to the reversible extinction of the intramolecular charge‐transfer character inherent to this type of dye. The main focus of the work is the covalent anchoring of the dyes to inert, porous polyacrylate beads so as to form a solid‐state sensor suitable for analysis of gases or flowing liquids. The final material is highly stable—its performance is undiminished after more than one year—and fully reversible over many cycles. The sensitivity is such that reactions can be followed by the naked eye and the detection limit is about 600 ppb for HCl and about 80 ppb for ammonia. Trace amounts of diphosgene can be detected, as can alkylating agents. The sensing action is indiscriminate and also operates when the beads are dispersed in aqueous media.