Ratiometric and Near‐Infrared Molecular Probes for the Detection and Imaging of Zinc Ions

The detection and imaging of Zn²⁺ in biological samples are of paramount interest owing to the role of this cation in physiological functions. This is possible only with molecular probes that specifically bind to Zn²⁺ and result in changes in emission properties. A “turn‐on” emission or shift in the emission color upon binding to Zn²⁺ should be ideal for in vivo imaging. In this context, ratiometric and near‐IR probes are of particular interest. Therefore, in the area of chemosensors or molecular probes, the design of fluorophores that allow ratiometric sensing or imaging in the near‐IR region is attracting the attention of chemists. The purpose of this Focus Review is to highlight recent developments in this area and stress the importance of further research for future 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.     

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.     

A New 3,5‐Bisporphyrinylpyridine Derivative as a Fluorescent Ratiometric Probe for Zinc Ions

A new 3,5‐disubstituted pyridine with two porphyrin moieties was prepared through an efficient synthetic approach involving 2‐formyl‐5,10,15,20‐tetraphenylporphyrin ( 1 ), piperidine, and catalytic amounts of [La(OTf)₃]. 3,5‐Bis(5,10,15,20‐tetraphenylporphyrin‐2‐ylmethyl)pyridine ( 2 ) was fully characterized and its sensing ability towards Zn²⁺, Cu²⁺, Hg²⁺, Cd²⁺, and Ag⁺ was evaluated in solution by absorption and fluorescence spectroscopy and in gas phase by using matrix‐assisted laser desorption/ionization (MALDI)‐TOF mass spectrometry. Strong changes in the ground and excited state were detected in the case of the soft metal ions Zn²⁺, Cd²⁺, Hg²⁺, and Cu²⁺. A three‐metal‐per‐ligand molar ratio was obtained in all cases and a significant ratiometric behavior was observed in the presence of Zn²⁺ with the appearance of a new band at 608 nm, which can be assigned to a metal‐to‐ligand charge transfer. The system was able to quantify 79 ppb of Zn²⁺ and the theoretical calculations are in accordance with the stoichiometry observed in solution. The gas‐phase sensorial ability of compound 2 towards all metal ions was confirmed by using MALDI‐TOF MS and in solid state by using polymeric films of polymethylmethacrylate (PMMA) doped with ligand 2 . The results showed that compound 2 can be analytically used to develop new colorimetric molecular devices that are able to discriminate between Hg²⁺ and Zn²⁺ in solid phase. The crystal structure of Zn^II complex of 3,5‐bisporphyrinylpyridine was unequivocally elucidated by using single‐crystal X‐ray diffraction studies.     

Mapping Live Cell Viscosity with an Aggregation‐Induced Emission Fluorogen by Means of Two‐Photon Fluorescence Lifetime Imaging

Intracellular viscosity is a crucial parameter that indicates the functioning of cells. In this work, we demonstrate the utility of TPE‐Cy, a cell‐permeable dye with aggregation‐induced emission (AIE) property, in mapping the viscosity inside live cells. Owing to the AIE characteristics, both the fluorescence intensity and lifetime of this dye are increased along with an increase in viscosity. Fluorescence lifetime imaging of live cells stained with TPE‐Cy reveals that the lifetime in lipid droplets is much shorter than that from the general cytoplasmic region. The loose packing of the lipids in a lipid droplet results in low viscosity and thus shorter lifetime of TPE‐Cy in this region. It demonstrates that the AIE dye could provide good resolution in intracellular viscosity sensing. This is also the first work in which AIE molecules are applied in fluorescence lifetime imaging and intracellular viscosity sensing.     

Photoelectrocyclization as an Activation Mechanism for Organelle‐Specific Live‐Cell Imaging Probes

Photoactivatable fluorophores are useful tools in live‐cell imaging owing to their potential for precise spatial and temporal control. In this report, a new photoactivatable organelle‐specific live‐cell imaging probe based on a 6π electrocyclization/oxidation mechanism is described. It is shown that this new probe is water‐soluble, non‐cytotoxic, cell‐permeable, and useful for mitochondrial imaging. The probe displays large Stokes shifts in both pre‐activated and activated forms, allowing simultaneous use with common dyes and fluorescent proteins. Sequential single‐cell activation experiments in dense cellular environments demonstrate high spatial precision and utility in single‐ or multi‐cell labeling experiments.     

FRET‐Based Mitochondria‐Targetable Dual‐Excitation Ratiometric Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells

Hydrogen sulfide (H₂S) is connected with various physiological and pathological functions. However, understanding the important functions of H₂S remains challenging, in part because of the lack of tools for detecting endogenous H₂S. Herein, compounds Ratio‐H₂S 1/2 are the first FRET‐based mitochondrial‐targetable dual‐excitation ratiometric fluorescent probes for H₂S on the basis of H₂S‐promoted thiolysis of dinitrophenyl ether. With the enhancement of H₂S concentration, the excitation peak at λ≈402 nm of the phenolate form of the hydroxycoumarin unit drastically increases, whereas the excitation band centered at λ≈570 nm from rhodamine stays constant and can serve as a reference signal. Thus, the ratios of fluorescence intensities at λ=402 and 570 nm (I₄₀₂/I₅₇₀) exhibit a drastic change from 0.048 in the absence of H₂S to 0.36 in the presence of 180 μM H₂S; this is a 7.5‐fold variation in the excitation ratios. The favorable properties of the probe include the donor and acceptor excitation bands, which exhibit large excitation separations (up to 168 nm separation) and comparable excitation intensities, high sensitivity and selectivity, and function well at physiological pH. In addition, it is demonstrated that the probe can localize in the mitochondria and determine H₂S in living cells. It is expected that this strategy will lead to the development of a wide range of mitochondria‐targetable dual‐excitation ratiometric probes for other analytes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.     

Ratiometric Single‐Nanoparticle Oxygen Sensors for Biological Imaging

It makes sense : Conjugated polymer nanoparticles doped with a platinum porphyrin dye exhibit bright phosphorescence that is highly sensitive to the concentration of molecular oxygen. The small size, extraordinary brightness, excellent sensitivity, and ratiometric emission, together with the demonstration of single‐particle sensing and cellular uptake, indicate the potential of the nanoparticle sensors for quantitative mapping of local molecular oxygen concentration.

     

A “Distorted‐BODIPY”‐Based Fluorescent Probe for Imaging of Cellular Viscosity in Live Cells

Cellular viscosity is a critical factor in governing diffusion‐mediated cellular processes and is linked to a number of diseases and pathologies. Fluorescent molecular rotors (FMRs) have recently been developed to determine viscosity in solutions or biological fluid. Herein, we report a “distorted‐BODIPY”‐based probe BV‐1 for cellular viscosity, which is different from the conventional “pure rotors”. In BV‐1 , the internal steric hindrance between the meso‐CHO group and the 1,7‐dimethyl group forced the boron–dipyrrin framework to be distorted, which mainly caused nonradiative deactivation in low‐viscosity environment. BV‐1 gave high sensitivity (x=0.62) together with stringent selectivity to viscosity, thus enabling viscosity mapping in live cells. Significantly, the increase of cytoplasmic viscosity during apoptosis was observed by BV‐1 in real time.     

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.     

Design of Ratiometric Fluorescent Probes Based on Arene–Metal‐Ion Interactions and Their Application to CdII and Hydrogen Sulfide Imaging in Living Cells

Non‐coordinative interactions between a metal ion and the aromatic ring of a fluorophore can act as a versatile sensing mechanism for the detection of metal ions with a large emission change of fluorophores. We report the design of fluorescent probes based on arene–metal‐ion interactions and their biological applications. This study found that various probes having different fluorophores and metal binding units displayed significant emission redshift upon complexation with metal ions, such as Ag^I, Cd^II, Hg^II, and Pb^II. X‐ray crystallography of the complexes confirmed that the metal ions were held in close proximity to the fluorophore to form an arene–metal‐ion interaction. Electronic structure calculations based on TDDFT offered a theoretical basis for the sensing mechanism, thus showing that metal ions electrostatically modulate the energy levels of the molecular orbitals of the fluorophore. A fluorescent probe was successfully applied to the ratiometric detection of the uptake of Cd^II ions and hydrogen sulfide (H₂S) in living cells. These results highlight the utility of interactions between arene groups and metal ions in biological analyses.     

Ubiquinone‐Rhodol (UQ‐Rh) for Fluorescence Imaging of NAD(P)H through Intracellular Activation

The nicotinamide adenine dinucleotide (NAD) derivatives NADH and NADPH are critical components of cellular energy metabolism and operate as electron carriers. A novel fluorescent ubiquinone‐rhodol derivative (UQ‐Rh) was developed as a probe for NAD(P)H. By using the artificial promoter [(η⁵‐C₅Me₅)Ir(phen)(H₂O)]²⁺, intracellular activation and imaging of NAD(P)H were successfully demonstrated. In contrast to bioorthogonal chemistry, this “bioparallel chemistry” approach involves interactions with native biological processes and could potentially be used to control or investigate cellular systems.     

Fluorescent Protein Capped Mesoporous Nanoparticles for Intracellular Drug Delivery and Imaging

A multifunctional system for intracellular drug delivery and simultaneous fluorescent imaging was constructed by using histidine‐tagged, cyan fluorescent protein (CFP)‐capped magnetic mesoporous silica nanoparticles (MMSNs). This protein‐capped multifunctional nanostructure is highly biocompatible and does not affect cell viability or proliferation. The CFP acts not only as a capping agent, but also as a fluorescent imaging agent. The nanoassembly was activated by histidine‐based replacement, leading to release of drug molecules encapsulated in the nanopores into the bulk solution. The fluorescent imaging functionality would allow noninvasive tracking of the nanoparticles in the body. By combining the drug delivery with cell‐imaging capability, these nanoparticles may provide valuable multifunctional nanoplatforms for biomedical applications.