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Photoactivatable derivatives of the bright and photostable Janelia Fluor dyes enable improved multicolor single-particle tracking and facile localization microscopy in cells. Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

NIR-II fluorophores have shown great promise for biomedical applications with superior in vivo optical properties. To date, few small-molecule NIR-II fluorophores have been discovered with donor-acceptor-donor (D-A-D) or symmetrical structures, and upconversion-mitochondria-targeted NIR-II dyes have not been reported. Herein, we report development of D-A type thiopyrylium-based NIR-II fluorophores with frequency upconversion luminescence (FUCL) at ~580 nm upon excitation at ~850 nm. H4-PEG-PT can not only quickly and effectively image mitochondria in live or fixed osteosarcoma cells with subcellular resolution at 1 nM, but also efficiently convert optical energy into heat, achieving mitochondria-targeted photothermal cancer therapy without ROS effects. H4-PEG-PT has been further evaluated in vivo and exhibited strong tumor uptake, specific NIR-II signals with high spatial and temporal resolution, and remarkable NIR-II image-guided photothermal therapy. This report presents the first D-A type thiopyrylium NIR-II theranostics for synchronous upconversion-mitochondria-targeted cell imaging, in vivo NIR-II osteosarcoma imaging and excellent photothermal efficiency. Currently available mitochondria-targeted fluorescent dyes emit only one color in the visible or NIR-I and their applications are limited. Here, the authors develop upconversion mitochondria-targeted NIR-II fluorophores for synchronous upconversion-mitochondria-targeted cell imaging, in vivo NIR-II osteosarcoma imaging and photothermal efficiency

Organic near-infrared room temperature phosphorescence materials have unparalleled advantages in bioimaging due to their excellent penetrability. However, limited by the energy gap law, the near-infrared phosphorescence materials (>650 nm) are very rare, moreover, the phosphorescence lifetimes of these materials are very short. In this work, we have obtained organic room temperature phosphorescence materials with long wavelengths (600/657–681/732 nm) and long lifetimes (102–324 ms) for the first time through the guest-host doped strategy. The guest molecule has sufficient conjugation to reduce the lowest triplet energy level and the host assists the guest in exciton transfer and inhibits the non-radiative transition of guest excitons. These materials exhibit good tissue penetration in bioimaging. Thanks to the characteristic of long lifetime and long wavelength emissive phosphorescence materials, the tumor imaging in living mice with a signal to background ratio value as high as 43 is successfully realized. This work provides a practical solution for the construction of organic phosphorescence materials with both long wavelengths and long lifetimes. Though room-temperature phosphorescence (RTP) in organics is advantageous for bioimaging, designing materials that meet lifetime and wavelength emission requirements is challenging. Here, the authors us a guest-host doped strategy to construct RTP materials with ultralong-lifetime, NIR emission.

A simple and general chemical structure change to a panel of cell-permeable small-molecule fluorophores increases their brightness and photostability, which will enable improved single-molecule studies and super-resolution imaging. Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N , N -dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

High-resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to substantial scattering and autofluorescence in tissue at visible (350–700 nm) and near-infrared (700–1,000 nm) wavelengths. Here, we enable real-time, non-invasive multicolour imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000–2,000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established relationships between structure and photophysical properties for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable near-infrared/SWIR excitation and single-channel detection, facilitating video-rate multicolour SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1,064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-colour imaging with high temporal and spatial resolutions.Conducting high-resolution, multiplexed imaging in living mammals is challenging because of considerable scattering and autofluorescence in tissue at visible and near-infrared wavelengths. Now, real-time, non-invasive multicolour imaging experiments in live animals have been achieved through the design of optical contrast agents for the shortwave infrared (SWIR, 1,000–2,000 nm) region and the introduction of excitation multiplexing with single-channel SWIR detection.

With suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in the 1,500- to 1,700-nm range of the near-IR (NIR) spectrum (NIR-IIb window) can afford high clarity and deep tissue penetration. However, there has been a lack of NIR-IIb fluorescent probes with sufficient brightness and aqueous stability. Here, we present a bright fluorescent probe emitting at ∼1,600 nm based on core/shell lead sulfide/cadmium sulfide (CdS) quantum dots (CSQDs) synthesized in organic phase. The CdS shell plays a critical role of protecting the lead sulfide (PbS) core from oxidation and retaining its bright fluorescence through the process of amphiphilic polymer coating and transferring to water needed for imparting aqueous stability and compatibility. The resulting CSQDs with a branched PEG outer layer exhibited a long blood circulation half-life of 7 hours and enabled through-skin, real-time imaging of blood flows in mouse vasculatures at an unprecedented 60 frames per second (fps) speed by detecting ∼1,600-nm fluorescence under 808-nm excitation. It also allowed through-skin in vivo confocal 3D imaging of tumor vasculatures in mice with an imaging depth of ∼1.2 mm. The PEG-CSQDs accumulated in tumor effectively through the enhanced permeation and retention effect, affording a high tumor-to-normal tissue ratio up to ∼32 owing to the bright ∼1,600-nm emission and nearly zero autofluorescence background resulting from a large ∼800-nm Stoke’s shift. The aqueous-compatible CSQDs are excreted through the biliary pathway without causing obvious toxicity effects, suggesting a useful class of ∼1,600-nm emitting probes for biomedical research.

Hydrogen sulfide (H ₂S) is a reactive small molecule generated in the body that can be beneficial or toxic owing to its potent redox activity. In living systems, disentangling the pathways responsible for H ₂S production and their physiological and pathological consequences remains a challenge in part due to a lack of methods for monitoring changes in endogenous H ₂S fluxes. The development of fluorescent probes with appropriate selectivity and sensitivity for monitoring production of H ₂S at biologically relevant signaling levels offers opportunities to explore its roles in a variety of systems. Here we report the design, synthesis, and application of a family of azide-based fluorescent H ₂S indicators, Sulfidefluor-4, Sulfidefluor-5 acetoxymethyl ester, and Sulfidefluor-7 acetoxymethyl ester, which offer the unique capability to image H ₂S generated at physiological signaling levels. These probes are optimized for cellular imaging and feature enhanced sensitivity and cellular retention compared with our previously reported molecules. In particular, Sulfidefluor-7 acetoxymethyl ester allows for direct, real-time visualization of endogenous H ₂S produced in live human umbilical vein endothelial cells upon stimulation with vascular endothelial growth factor (VEGF). Moreover, we show that H ₂S production is dependent on NADPH oxidase–derived hydrogen peroxide (H ₂O ₂), which attenuates VEGF receptor 2 phosphorylation and establishes a link for H ₂S/H ₂O ₂ crosstalk.

Carbon dots (CDs) have a wide range of applications in chemical, physical and biomedical research fields. We are particularly interested in the use of CDs as fluorescence nanomaterials for targeted tumor cell imaging. One of the important aspects of success is to enhance the fluorescence quantum yields (QY) of CDs as well as increase their targetability to tumor cells. However, most of the reported CDs are limited by relative low QY. In the current study, for the first time, one-step synthesis of highly luminescent CDs by using folic acid (FA) as single precursor was obtained in natural water through hydrothermal method. The as-prepared CDs exhibited QY as high as 94.5% in water, which is even higher than most of organic fluorescent dyes. The obtained CDs showed excellent photoluminescent activity, high photostability and favorable biocompatibility. The FA residuals in CDs led to extraordinary targetability to cancer cells and promoted folate receptor-mediated cellular uptake successfully, which holds a great potential in biological and bioimaging studies.

Fluorescence imaging in near-infrared IIb (NIR-IIb, 1500–1700 nm) spectrum holds a great promise for tissue imaging. While few inorganic NIR-IIb fluorescent probes have been reported, their organic counterparts are still rarely developed, possibly due to the shortage of efficient materials with long emission wavelength. Herein, we propose a molecular design philosophy to explore pure organic NIR-IIb fluorophores by manipulation of the effects of twisted intramolecular charge transfer and aggregation-induced emission at the molecular and morphological levels. An organic fluorescent dye emitting up to 1600 nm with a quantum yield of 11.5% in the NIR-II region is developed. NIR-IIb fluorescence imaging of blood vessels and deeply-located intestinal tract of live mice based on organic dyes is achieved with high clarity and enhanced signal-to-background ratio. We hope this study will inspire further development on the evolution of pure organic NIR-IIb dyes for bio-imaging. Light has limited penetration depth in vivo which has led to increasing interest in NIR-II fluorophores. Here, the authors report on the design and testing of an organic aggregation induced emission fluorophore with high quantum yield and demonstrate imaging of vasculature and intestines in live mice.

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted ‘Janelia Fluor’ (JF) dyes useful for biological imaging experiments in cells and in vivo. A general tuning strategy is introduced for improving the utility of rhodamines for biological imaging applications. The strategy yielded bright, versatile and bioavailable far-red and near-infrared ‘Janelia Fluor’ dyes.