A Systematic Study of Coumarin–Tetrazine Light-Up Probes for Bioorthogonal Fluorescence Imaging

Fluorescent probes that light-up upon reaction with complementary bioorthogonal reagents are superior tools for no-wash fluorogenic bioimaging applications. In this work, a thorough study is presented on a set of seventeen structurally diverse coumarin–tetrazine probes that produce fluorescent dyes with exceptional turn-on ratios when reacted with trans-cyclooctene (TCO) and bicyclononyne (BCN) dienophiles. In general, formation of the fully aromatic pyridazine-containing dyes resulting from the reaction with BCN was found superior in terms of fluorogenicity. However, evaluation of the probes in cellular imaging experiments revealed that other factors, such as reaction kinetics and good cell permeability, prevail over the fluorescence turn-on properties. The best compound identified in this study showed excellent performance in live cell-labeling experiments and enabled no-wash fluorogenic imaging on a timescale of seconds.     

Ultrafluorogenic Monochromophore-Type BODIPY-Tetrazine Series for Dual-Color Bioorthogonal Imaging with a Single Probe

Various fluorogenic probes utilizing tetrazine (Tz) as a fluorescence quencher and bioorthogonal reaction partner have been extensively studied over the past few decades. Herein, we synthesized a series of boron-dipyrromethene (BODIPY)-Tz probes using monochromophoric design strategy for bioorthogonal cellular imaging. The BODIPY-Tz probes exhibited excellent bicyclo[6.1.0]nonyne (BCN)-selective fluorogenicity with three- to four-digit-fold enhancements in fluorescence over a wide range of emission wavelengths, including the far-red region. Furthermore, we demonstrated the applicability of BODIPY-Tz probes in bioorthogonal fluorescence imaging of cellular organelles without washing steps. We also elucidated the aromatized pyridazine moiety as the origin of BCN-selective fluorogenic behavior. Additionally, we discovered that the fluorescence of the trans-cyclooctene (TCO) adducts was quenched in aqueous media via photoinduced electron transfer (PeT) process. Interestingly, we observed a distinctive recovery of the initially quenched fluorescence of BODIPY-Tz-TCO upon exposure to hydrophobic media, accompanied by a significant bathochromic shift of its emission wavelength relative to that exhibited by the corresponding BODIPY-Tz-BCN. Leveraging this finding, for the first time, we achieved dual-color bioorthogonal cellular imaging with a single BODIPY-Tz probe.     

A general strategy for in situ assembly of light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling

Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer (ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction. By introducing iodo group at the appropriate position, five fluorophores with different scaffolds including naphthalimide, coumarin, naphthalene sulfonate, nitrobenzoxadiazole, and acetonaphthone, were designed as bioorthogonal multicolor fluorogenic probes, which could produce significant fluorescence enhancement and high fluorescence quantum yield after Suzuki-Miyaura reaction with aryl boronic acid or boronate. Manipulating the substituents and π scaffold in the fluorophores allows fine-tuning of their photophysical properties. With this strategy, we succeeded in peptide conjugation, no-wash fluorogenic protein labeling, and mitochondria-selective bioorthogonal imaging in live cells.     

Ultrafluorogenic Coumarin–Tetrazine Probes for Real‐Time Biological Imaging

We have developed a series of new ultrafluorogenic probes in the blue‐green region of the visible‐light spectrum that display fluorescence enhancement exceeding 11 000‐fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans‐cyclooctene(TCO)–tetrazine chemistry platform. By exploiting highly efficient through‐bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No‐wash, fluorogenic imaging of diverse targets including cell‐surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.     

Two-photon fluorescence microscopy imaging of cellular oxidative stress using profluorescent nitroxides

A range of varying chromophore nitroxide free radicals and their nonradical methoxyamine analogues were synthesized and their linear photophysical properties examined. The presence of the proximate free radical masks the chromophore's usual fluorescence emission, and these species are described as profluorescent. Two nitroxides incorporating anthracene and fluorescein chromophores (compounds 7 and 19, respectively) exhibited two-photon absorption (2PA) cross sections of approximately 400 G.M. when excited at wavelengths greater than 800 nm. Both of these profluorescent nitroxides demonstrated low cytotoxicity toward Chinese hamster ovary (CHO) cells. Imaging colocalization experiments with the commercially available CellROX Deep Red oxidative stress monitor demonstrated good cellular uptake of the nitroxide probes. Sensitivity of the nitroxide probes to H(2)O(2)-induced damage was also demonstrated by both one- and two-photon fluorescence microscopy. These profluorescent nitroxide probes are potentially powerful tools for imaging oxidative stress in biological systems, and they essentially "light up" in the presence of certain species generated from oxidative stress. The high ratio of the fluorescence quantum yield between the profluorescent nitroxide species and their nonradical adducts provides the sensitivity required for measuring a range of cellular redox environments. Furthermore, their reasonable 2PA cross sections provide for the option of using two-photon fluorescence microscopy, which circumvents commonly encountered disadvantages associated with one-photon imaging such as photobleaching and poor tissue penetration.     

A two-photon fluorescent probe with a large turn-on signal for imaging hydrogen sulfide in living tissues

A two-photon fluorescence turn-on H₂S probe GCTPOC–H₂S based on a two-photon platform with a large cross-section, GCTPOC, and a sensitive H₂S recognition site, dinitrophenyl ether was constructed. The probe GCTPOC–H₂S exhibits desirable properties such as high sensitivity, high selectivity, functioning well at physiological pH and low cytotoxicity. In particular, the probe shows a 120-fold enhancement in the presence of Na₂S (500 μM), which is larger than the reported two-photon fluorescent H₂S probes. The large fluorescence enhancement of the two-photon probe GCTPOC–H₂S renders it attractive for imaging H₂S in living tissues with deep tissue penetration. Significantly, we have demonstrated that the probe GCTPOC–H₂S is suitable for fluorescence imaging of H₂S in living tissues with deep penetration by using two-photon microscopy. The further application of the two-photon probe for the investigation of biological functions and pathological roles of H₂S in living systems is under progress.     

Organelle-specific detection of phosphatase activities with two-photon fluorogenic probes in cells and tissues

Two-photon fluorescence microscopy (TPFM) provides key advantages over conventional fluorescence imaging techniques, namely, increased penetration depth, lower tissue autofluorescence and self-absorption, and reduced photodamage and photobleaching and therefore is particularly useful for imaging deep tissues and animals. Enzyme-detecting, small molecule probes provide powerful alternatives over conventional fluorescent protein (FP)-based methods in bioimaging, primarily due to their favorable photophysical properties, cell permeability, and chemical tractability. In this article, we report the first fluorogenic, small molecule reporter system (Y2/Y1) capable of imaging endogenous phosphatase activities in both live mammalian cells and Drosophila brains. The one- and two-photon excited photophysical properties of the system were thoroughly investigated, thus confirming the system was indeed a suitable Turn-ON fluorescence pair for TPFM. To our knowledge, this is the first enzyme reporting two-photon fluorescence bioimaging system which was designed exclusively from a centrosymmetric dye possessing desirable two-photon properties. By conjugation of our reporter system to different cell-penetrating peptides (CPPs), we were able to achieve organelle- and tumor cell-specific imaging of phosphatase activities with good spatial and temporal resolution. The diffusion problem typically associated with most small molecule imaging probes was effectively abrogated. We further demonstrated this novel two-photon system could be used for imaging endogenous phosphatase activities in Drosophila brains with a detection depth of >100 μm.     

Molecular Design of d-Luciferin-Based Bioluminescence and 1,2-Dioxetane-Based Chemiluminescence Substrates for Altered Output Wavelength and Detecting Various Molecules

Optical imaging including fluorescence and luminescence is the most popular method for the in vivo imaging in mice. Luminescence imaging is considered to be superior to fluorescence imaging due to the lack of both autofluorescence and the scattering of excitation light. To date, various luciferin analogs and bioluminescence probes have been developed for deep tissue and molecular imaging. Recently, chemiluminescence probes have been developed based on a 1,2-dioxetane scaffold. In this review, the accumulated findings of numerous studies and the design strategies of bioluminescence and chemiluminescence imaging reagents are summarized.     

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click-to-Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse-electron-demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans-cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single-walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO-caged molecule was used to deliver active effector molecules. To optimize a turn-on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near-infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real-time, non-invasive tumour visualization with a high target-to-background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine-functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off-site activation of fluorophore/drug.     

Polyfluorene based conjugated polymer nanoparticles for two-photon live cell imaging

Two-photon excitation microscopy (2PEM) has been known as a noninvasive and powerful bio-imaging tool for studying living cells, intact tissues and living animals because of their unique advantages such as localized excitation, deep tissue penetration as well as less photo-damage. However, the major limitations that hinder its practical applications in biological systems are low two-photon absorption cross sections of conventional fluorescence probes. Conjugated polymer nanoparticles (CPNs) consisting of highly fluorescent conjugated polymers are promising fluorescent probes for 2PEM due to their unique advantages including large two-photon absorption cross sections, high fluorescence quantum yield, good photo-stability and biocompatibility, facile chemical synthesis, tunable optical properties as well as versatile surface modifications. This account summarizes the recent efforts of our group on development of novel polyfluorene based CPNs as 2PEM contrast agents for live cell imaging.     

Multicolor, one- and two-photon imaging of enzymatic activities in live cells with fluorescently Quenched Activity-Based Probes (qABPs)

Fluorescence imaging provides an indispensable way to locate and monitor biological targets within complex and dynamic intracellular environments. Of the various imaging agents currently available, small molecule-based probes provide a powerful tool for live cell imaging, primarily due to their desirable properties, including cell permeability (as a result of their smaller sizes), chemical tractability (e.g., different molecular structures/designs can be installed), and amenability to imaging a wide variety of biological events. With a few exceptions, most existing small molecule probes are however not suitable for in vivo bioimaging experiments in which high-resolution studies of enzyme activity and localization are necessary. In this article, we reported a new class of fluorescently Quenched Activity-Based Probes (qABPs) which are highly modular, and can sensitively image (through multiple enzyme turnovers leading to fluorescence signal amplification) different types of enzyme activities in live mammalian cells with good spatial and temporal resolution. We have also incorporated two-photon dyes into our modular probe design, enabling for the first time activity-based, fluorogenic two-photon imaging of enzyme activities. This, hence, expands the repertoire of 'smart', responsive probes currently available for live cell bioimaging experiments.     

AIEgen based light-up probes for live cell imaging

Fluorescent light-up probes comprising a tetraphenylethene unit with aggregation-induced emission (AIE) characteristics and a water-soluble peptide have been designed and synthesized which provide cell membrane and nuclear permeability to live cells. This strategy has offered new opportunities for the development of probes with light-up ability and good signal-to-noise ratio. The selectivity or targeting specificity is determined by the peptide sequence, i.e. a nuclear localization signal that leads to nucleus imaging and a cell biomarker targeting peptide that offers specific light-up imaging of HT-29 cells.     

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click‐to‐Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse‐electron‐demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans‐cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single‐walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO‐caged molecule was used to deliver active effector molecules. To optimize a turn‐on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near‐infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real‐time, non‐invasive tumour visualization with a high target‐to‐background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine‐functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off‐site activation of fluorophore/drug.     

Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy

A novel method for the synthesis of highly monodispersed hydrophillic InP-ZnS nanocrystals and their use as luminescence probes for live cell imaging is reported. Hydrophobic InP-ZnS nanocrystals are prepared by a new method that yields high-quality, luminescent core-shell nanocrystals within 6-8 h of total reaction time. Then by carefully manipulating the surface of these passivated nanocrystals, aqueous dispersions of folate-conjugated nanocrystals (folate-QDs) with high photostability are prepared. By use of confocal microscopy, we demonstrate the receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as KB cells. These folate-QDs tend to accumulate in multi-vescicular bodies of KB cells after 6 h of incubation. Receptor-mediated delivery was confirmed by comparison with the uptake of these particles in folate-receptor-negative cell lines such as A549. Efficient two-photon excitation of these particles and two-photon imaging using these particles are also demonstrated. The use of these InP-ZnS nanoparticles and their efficient two-photon excitation can be potentially useful for deep tissue imaging for future in vivo studies.     

Multivalent Siderophore–DOTAM Conjugates as Theranostics for Imaging and Treatment of Bacterial Infections

There is a strong need to better diagnose infections at deep body sites through noninvasive molecular imaging methods. Herein, we describe the synthesis and characterization of probes based on siderophore conjugates with catechol moieties and a central DOTAM scaffold. The probes can accommodate a metal ion as well as an antibiotic moiety and are therefore suited for theranostic purposes. The translocation of the conjugates across the outer and inner cell membranes of E. coli was confirmed by growth recovery experiments with enterobactin-deficient strains, by the antibacterial activity of ampicillin conjugates, and by confocal imaging using a fluorogen-activating protein–malachite green system adapted to E. coli. The suitability of the probes for in vivo imaging was demonstrated with a Cy5.5 conjugate in mice infected with P. aeruginosa.     

Recent advances in organic-dye-based photoacoustic probes for biosensing and bioimaging

Photoacoustic imaging(PAI) is a non-destructive biomedical imaging technology with broad application prospects. PAI combines the advantages of optical imaging and ultrasound imaging with high selectivity and deep penetration to overcome the high scattering limitation of light in tissues. This emerging technology also achieves high-resolution and high-contrast imaging of deep tissue in vivo. Recently, photoacoustic(PA) probes based on organic dyes have emerged prominently in biosensing and bioimaging due to their excellent optical properties and structural adaptability. This paper gives an outline of the basic PAI principles and focuses on the application of organic-dye-based PA probes for molecular detection and in vivo imaging. The advantages of PAI technology and the drawbacks of current PA probes are then summarized. Finally, the prospects for application are evaluated considering the potential challenges in the biomedical fields.     

Activatable Molecular Probes for Second Near‐Infrared Fluorescence,Chemiluminescence, and Photoacoustic Imaging

Optical imaging plays a crucial role in biomedicine. However, due to strong light scattering and autofluorescence in biological tissue between 650–900 nm, conventional optical imaging often has a poor signal‐to‐background ratio and shallow penetration depth, which limits its ability in deep‐tissue in vivo imaging. Second near‐infrared fluorescence, chemiluminescence, and photoacoustic imaging modalities mitigate these issues by their respective advantages of minimized light scattering, eliminated external excitation, and ultrasound detection. To enable disease detection, activatable molecular probes (AMPs) with the ability to change their second near‐infrared fluorescence, chemiluminescence, or photoacoustic signals in response to a biomarker have been developed. This Minireview summarizes the molecular design strategies, sensing mechanisms, and imaging applications of AMPs. The potential challenges and perspectives of AMPs in deep‐tissue imaging are also discussed.     

Emission ratiometric imaging of intracellular zinc: design of a benzoxazole fluorescent sensor and its application in two-photon microscopy

Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and properties of the benzoxazole-based, ratiometric zinc probe, Zinbo-5. This bright fluorescent reporter has a quantum yield of 0.1 in the zinc-form, exhibits a Kd for Zn2+ in the nanomolar range, and shows significant changes in both excitation and emission maxima upon zinc binding. The utility of this cell permeable probe is demonstrated in fluorescence microscopy emission ratio imaging experiments on mammalian cells. We further show that Zinbo-5 is well suited for two-photon excitation microscopy ratio imaging and can readily reveal changes in intracellular zinc concentration within optical planes of single cells. To the best of our knowledge, this is the first example of two-photon excitation microscopy applied to ratio imaging of zinc. These methods can be applied to real-time emission or excitation ratio imaging studies of zinc physiology in living cells.     

A cyanine based fluorophore emitting both single photon near-infrared fluorescence and two-photon deep red fluorescence in aqueous solution

Optical imaging provides an indispensable way to locate tumors in their early stages with high sensitivity and signal to background ratio. A heptamethine cyanine based fluorophore that emits both single photon near-infrared fluorescence and two-photon deep red fluorescence under physiological conditions was developed. Linear and nonlinear photophysical properties of this fluorophore were investigated and it demonstrated the capability to label lysosomes in cancer cells. The advantages of this fluorophore, including tolerable cytotoxicity, high fluorescence quantum yield, and the ability to emit both near-infrared single photon fluorescence and deep red two photon fluorescence in aqueous solution, give it potential to be used in intra-operatively optical image-guided tumor excision followed by two-photon fluorescence microscopy biopsy analysis after a single administration.     

双光子荧光探针研究及其应用*

双光子荧光显微成像兼具诸如近红外激发、暗场成像、避免荧光漂白和光致毒、定靶激发、高横向分辨率与纵向分辨率、降低生物组织吸光系数及降低组织自发荧光干扰等特点而显著地优于单光子荧光显微成像,为生命科学研究提供了更为锐利的工具。而用于像离子的含量及其对生理的影响、离子参与的生理活动机制、离子与分子的作用、特定分子的分布及其相互作用等方面研究的双光子荧光探针,是实现成像的关键。双光子荧光探针的研究旨在促进双光子荧光显微镜应用的发展,促进生命科学、医学科学的快速发展,同时也带动双光子荧光探针所隶属的化学这一学科的发展。因此对双光子荧光探针的研究具有重要的理论和实践意义。该文综述了双光子荧光显微成像的优点、双光子荧光探针设计的原理及双光子荧光探针在离子分析方面的应用,并展望了这类荧光探针的发展趋势与应用前景。