Superresolution imaging of targeted proteins in fixed and living cells using photoactivatable organic fluorophores

Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.     

Regiospecific synthesis of 1,2-disubstituted (hetero)aryl fused imidazoles with tunable fluorescent emission

A palladium-catalyzed two or fourfold amination was established that allows regiospecific synthesis of a diversity-oriented library of 1,2-disubstituted (hetero)aryl fused imidazoles, and provides an exceptional tool for the discovery of fluorescent scaffolds with tunable fluorescence emission. These fluorophores have been applied as fluorescent probes for live cell imaging.     

Overview of Syntheses and Molecular-Design Strategies for Tetrazine-Based Fluorogenic Probes

Various bioorthogonal chemistries have been used for fluorescent imaging owing to the advantageous reactions they employ. Recent advances in bioorthogonal chemistry have revolutionized labeling strategies for fluorescence imaging, with inverse electron demand Diels–Alder (iEDDA) reactions in particular attracting recent attention owing to their fast kinetics and excellent specificity. One of the most interesting features of the iEDDA labeling strategy is that tetrazine-functionalized dyes are known to act as fluorogenic probes. In this review, we will focus on the synthesis, molecular-design strategies, and bioimaging applications of tetrazine-functionalized fluorogenic probes. Traditional Pinner reaction and “Pinner-like” reactions for tetrazine synthesis are discussed here, as well as metal-catalyzed C–C bond formations with convenient tetrazine intermediates and the fabrication of tetrazine-conjugated fluorophores. In addition, four different quenching mechanisms for tetrazine-modified fluorophores are presented.     

Fluorescent Organic Small Molecule Probes for Bioimaging and Detection Applications

The activity levels of key substances (metal ions, reactive oxygen species, reactive nitrogen, biological small molecules, etc.) in organisms are closely related to intracellular redox reactions, disease occurrence and treatment, as well as drug absorption and distribution. Fluorescence imaging technology provides a visual tool for medicine, showing great potential in the fields of molecular biology, cellular immunology and oncology. In recent years, organic fluorescent probes have attracted much attention in the bioanalytical field. Among various organic fluorescent probes, fluorescent organic small molecule probes (FOSMPs) have become a research hotspot due to their excellent physicochemical properties, such as good photostability, high spatial and temporal resolution, as well as excellent biocompatibility. FOSMPs have proved to be suitable for in vivo bioimaging and detection. On the basis of the introduction of several primary fluorescence mechanisms, the latest progress of FOSMPs in the applications of bioimaging and detection is comprehensively reviewed. Following this, the preparation and application of fluorescent organic nanoparticles (FONPs) that are designed with FOSMPs as fluorophores are overviewed. Additionally, the prospects of FOSMPs in bioimaging and detection are discussed.     

Development of Small-Molecule Fluorescent Probes Targeting Enzymes

As biological catalysts, enzymes are vital in controlling numerous metabolic reactions. The regulation of enzymes in living cells and the amount present are indicators of the metabolic status of cell, whether in normal condition or disease. The small-molecule fluorescent probes are of interest because of their high sensitivity and selectivity, as well as their potential for automated detection. Fluorescent probes have been useful in targeting particular enzymes of interest such as proteases and caspases. However, it is difficult to develop an ideal fluorescent probe for versatile purposes. In the future, the design and synthesis of enzyme-targeting fluorescent probes will focus more on improving the selectivity, sensitivity, penetration ability and to couple the fluorescent probes with other available imaging molecules/technologies.     

Fluorescent Probes Based on Nucleophilic Substitution–Cyclization for Hydrogen Sulfide Detection and Bioimaging

The design, synthesis, properties, and cell imaging applications of a series of 2‐pyridyl disulfide based fluorescent probes (WSP1, WSP2, WSP3, WSP4 and WSP5) for hydrogen sulfide detection are reported. The strategy is based on the dual‐nucleophilicity of hydrogen sulfide. A hydrogen sulfide mediated tandem nucleophilic substitution‐cyclization reaction is used to release the fluorophores and turn on the fluorescence. The probes showed high sensitivity and selectivity for hydrogen sulfide over other reactive sulfur species, including cysteine and glutathione.     

Chemically induced photoswitching of fluorescent probes--a general concept for super-resolution microscopy

We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.     

Fluorescent hybridization probes for nucleic acid detection

Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.     

Strategies for Tumor Hypoxia Imaging Based on Aggregation-Induced Emission Fluorogens

Hypoxia, as a crucial characteristic of cancer, has become an extremely significant direction for researchers to construct fluorescent probes for early diagnosis of tumors. Aggregation-induced emission fluorogens (AIEgens) possess many superior properties to those of conventional fluorophores due to aggregation-induced emission (AIE) features, such as a linear concentration-dependent increase in brightness, remarkable resistance to photobleaching, and the long-term tracking and imaging of cells. Constructing hypoxic response AIEgen-based probes will be very useful for the early diagnosis of tumors. Herein, several hypoxia-responsive probes based on AIEgens reported in the last three years are reported; these examples may lead to the construction of hypoxia-responsive AIE probes used for tumor hypoxia imaging in the future. In addition, typical, conventional hypoxia-responsive bioprobes are presented to further understand hypoxia-responsive fluorescent probes based on AIEgens.     

Interfacing Click Chemistry with Automated Oligonucleotide Synthesis for the Preparation of Fluorescent DNA Probes Containing Internal Xanthene and Cyanine Dyes

Double‐labeled oligonucleotide probes containing fluorophores interacting by energy‐transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2′‐O‐propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid‐phase copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy‐transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40–110 nm), quenched fluorescence of single‐stranded probes accompanied by up to 7.7‐fold light‐up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single‐nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM ).     

Nanometer fluorescent hybrid silica particle as ultrasensitive and photostable biological labels

Nanometer-sized fluorescent hybrid silica (NFHS) particles were prepared for use as sensitive and photostable fluorescent probes in biological staining and diagnostics. The first step of the synthesis involves the covalent modification of 3-aminopropyltrimethoxysilane with an organic fluorophore, such as fluorescein isothiocyanate, under N2 atmosphere for getting a fluorescent silica precursor. Then the NFHS particles, with a diameter of well below 40 nm, were prepared by controlled hydrolysis of the fluorescent silica precursor with tetramethoxysilane (TMOS) using the reverse micelle technique. The fluorophores are dispersed homogeneously in the silica network of the NFHS particles and well protected from the environmental oxygen. Furthermore, since the fluorophores are covalently bound to the silica network, there is no migration, aggregation and leakage of the fluorophores. In comparison with common single organic fluorophores, these particle probes are brighter, more stable against photobleaching and do not suffer from intermittent on/off light emission (blinking). We have used these newly developed NFHS particles as a fluorescent marker to label antibodies, using silica immobilization method, for the immunoassay of human alpha-fetoprotein (AFP). The detection limit of this method was down to 0.05 ng mL(-1) under our current experimental conditions. We think this material would attract much attention and be applied widely in biotechnology.     

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).     

荧光素类荧光分子探针

荧光素衍生物是重要的荧光探针,在检测和生物成像等领域中显示出巨大的前景。因此,急需对功能性荧光素结构探针的设计策略进行深入研究。通常通过引入醛基或酯化到荧光素呫吨环和苯部分来构建探针,由于其高活性,这些衍生物可以与分析物复合以发生颜色和荧光强度的变化。本文总结了荧光素的修饰位点及方法,介绍了荧光素探针的合成、性质及应用,并对近五年荧光素探针对不同分析物(包括金属阳离子、阴离子、小分子和生物大分子)的检测进行分类说明,旨在为高灵敏度荧光素探针的筛选和生物检测提供参考,并推动其在分析物传感和检测中的进一步应用。     

Functional Near‐Infrared Fluorescent Probes

Near‐infrared (NIR) fluorescent probes have attracted much attention, but despite the availability of various NIR fluorophores, only a few functional NIR probes, that is, probes whose absorption and/or fluorescence spectra change upon specific reaction with biomolecules, have been developed. However, functional probes operating in the NIR range that can be targeted to protons, metal ions, nitric oxide, β‐galactosidase, and cellular stress markers are expected to be effective for fluorescence imaging in vivo. This Focus Review concentrates on these functional NIR probes themselves, not their applications.     

Amino(oligo)thiophene-based environmentally sensitive biomembrane chromophores

There is a growing need for cellular imaging with fluorescent probes that emit at longer wavelengths to minimize the effects of absorption, autofluorescence, and scattering from biological tissue. In this paper a series of new environmentally sensitive hemicyanine dyes featuring amino(oligo)thiophene donors have been synthesized via aldol condensation between a 4-methylpyridinium salt and various amino(oligo)thiophene carboxaldehydes, which were, in turn, obtained from amination of bromo(oligo)thiophene carboxaldehyde. Side chains on these fluorophores impart a strong affinity for biological membranes. Compared with benzene analogues, these thiophene fluorophores show significant red shift in the absorption and emission spectra, offering compact red and near-infrared emitting fluorophores. More importantly, both the fluorescence quantum yields and the emission peaks are very sensitive to various environmental factors such as solvent polarity or viscosity, membrane potential, and membrane composition. These chromophores also exhibit strong nonlinear optical properties, including two-photon fluorescence and second harmonic generation, which are themselves environmentally sensitive. The combination of long wavelength fluorescence and nonlinear optical properties make these chromophores very suitable for applications that require sensing or imaging deep inside tissues.     

Magnesium azaphthalocyanines: an emerging family of excellent red-emitting fluorophores

Magnesium(II), zinc(II), and metal-free phthalocyanines (Pcs) and azaphthalocyanines (AzaPcs) containing alkylsulfanyl, aryloxy, and dialkylamino peripheral substituents have been synthesized. The complexation of magnesium(II) by metal-free Pcs and AzaPcs has been studied in detail to determine the optimal reaction conditions necessary to ensure a complete conversion. Photophysical and photochemical measurements in tetrahydrofuran showed that magnesium(II) AzaPcs with aryloxy and alkylsulfanyl substituents have excellent fluorescent properties (Φ(F) up to 0.73) and that the corresponding zinc(II) Pcs are efficient singlet oxygen producers (Φ(Δ) up to 0.68). The presence of dialkylamino substituents causes intramolecular charge transfer within the molecule that competes with fluorescence and singlet oxygen formation. Alkylsulfanyl MgAzaPc and ZnAzaPc were the most photostable compounds among the series of studied derivatives. In addition, high molar absorption coefficients (ε ～ 300,000 M(-1) cm(-1)), absorption (λ(max) ～ 650 nm), and emission (λ(em) ～ 660 nm, high Φ(F)) in the red region suggest that these molecules are potential fluorescent probes that are superior to the commercial red cyanine dye Cy5. MgAzaPc, when incorporated into lipidic bilayers of liposomes, maintains excellent fluorescence properties (Φ(F) = 0.64). Water-soluble MgAzaPc with quaternary ammonium peripheral substituents retained a high fluorescence quantum yield even in water (Φ(F) = 0.25). The described properties show that magnesium(II) AzaPcs are excellent red-emitting fluorophores with potential applications as fluorescent probes in sensing or in vitro imaging applications.     

Single‐Molecule Spectroscopy,Imaging, and Photocontrol: Foundations for Super‐Resolution Microscopy (Nobel Lecture)

The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single‐molecule limit in 1989 using frequency‐modulation laser spectroscopy. In the early 90s, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later super‐resolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super‐resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized.     

Systematic Exploration of Furoindolizine-Based Molecular Frameworks towards a Versatile Fluorescent Platform

The photophysical behaviors of fluorescent molecules largely determine their major utility in biological studies. Despite their well-defined characteristics, classical fluorophores have often been challenged by their limited synthetic methodology and tunability in adjusting intrinsic optical properties. A novel heterocyclic core equipped with modular functional groups could offer the flexibility to control its photophysical properties with a minimum synthetic effort. By conducting a systematic analysis guided by quantum calculations, we proposed the furoindolizine-based molecular framework as a unique fluorescent platform capable of providing versatile photophysical properties with minimal structural modification. A broad tunability of furoindolizine derivatives′ photophysical properties such as emission wavelength, Stokes shift, fluorescent brightness, and charge transfer characteristics was achieved through synergistic interaction between two functional moieties. Furthermore, this modular platform led to live-cell imaging probes with two distinct optical features simply by reorganizing a pair of functional moieties.     

Unravelling the effect of temperature on viscosity-sensitive fluorescent molecular rotors

Viscosity and temperature variations in the microscopic world are of paramount importance for diffusion and reactions. Consequently, a plethora of fluorescent probes have evolved over the years to enable fluorescent imaging of both parameters in biological cells. However, the simultaneous effect of both temperature and viscosity on the photophysical behavior of fluorophores is rarely considered, yet unavoidable variations in temperature can lead to significant errors in the readout of viscosity and vice versa. Here we examine the effect of temperature on the photophysical behavior of three classes of viscosity-sensitive fluorophores termed ‘molecular rotors’. For each of the fluorophores we decouple the effect of temperature from the effect of viscosity. In the case of the conjugated porphyrin dimer, we demonstrate that, uniquely, simultaneous dual-mode lifetime and intensity measurements of this fluorophore can be used for measuring both viscosity and temperature concurrently.     

Combinatorial discovery of full-color-tunable emissive fluorescent probes using a single core skeleton, 1,2-dihydropyrrolo[3,4-beta]indolizin-3-one

We developed a novel fluorescent core skeleton, 1,2-dihydropyrrolo[3,4-beta]indolizin-3-one, by complexity-generating one-pot reactions through 1,3-dipolar cyclization followed by oxidative aromatization. This fluorescent core skeleton can accommodate various wavelengths of emission maxima by changing the electronic properties of substituents, which was postulated by computational studies. The full-color-tunable emission maxima were achieved with a single core skeleton by changing the substituents using the combinatorial approach. These novel fluorophores have excellent photophysical and photochemical properties: moderate to excellent quantum yields, resistance to the photobleaching, pH-independent fluorescence, large Stokes shifts, druglike lipophilicity for membrane permeability, etc. Further, we successfully demonstrated the bioapplication of fluorophores B1 and B5 in the immunofluorescence for visualizing cellular compartments of HeLa cells.