2X-Rhodamine: A Bright and Fluorogenic Scaffold for Developing Near-Infrared Chemigenetic Indicators.

Chemigenetic fusion of synthetic dyes with genetically encoded protein tags presents a promising avenue for in vivo imaging. However, its full potential has been hindered by the lack of bright and fluorogenic dyes operating in the "tissue transparency" near-infrared window (NIR, 700-1700 nm). Here, we report 2X-rhodamine (2XR), a novel bright scaffold that allows for the development of live-cell-compatible, NIR-excited variants with strong fluorogenicity beyond 1000 nm. 2XR utilizes a rigidified π-skeleton featuring dual atomic bridges and functions via a spiro-based fluorogenic mechanism. This design affords longer wavelengths, higher quantum yield (ΦF = 0.11), and enhanced fluorogenicity in water when compared to the phosphine oxide-cored, or sulfone-cored rhodamine, the NIR fluorogenic benchmarks currently used. We showcase their bright performance in video-rate dynamic imaging and targeted deep-tissue molecular imaging in vivo. Notably, we develop a 2XR variant, 2XR715-HTL, an NIR fluorogenic ligand for the HaloTag protein, enabling NIR genetically encoded calcium sensing and the first demonstration of in vivo chemigenetic labeling beyond 1000 nm. Our work expands the library of NIR fluorogenic tools, paving the way for in vivo imaging and sensing with the chemigenetic approach.

Ultra-photostable small-molecule dyes facilitate near-infrared biophotonics.

Long-wavelength, near-infrared small-molecule dyes are attractive in biophotonics. Conventionally, they rely on expanded aromatic structures for redshift, which comes at the cost of application performance such as photostability, cell permeability, and functionality. Here, we report a ground-state antiaromatic strategy and showcase the concise synthesis of 14 cationic aminofluorene dyes with mini structures (molecular weights: 299-504 Da) and distinct spectra covering 700-1600 nm. Aminofluorene dyes are cell-permeable and achieve rapid renal clearance via a simple 44 Da carboxylation. This accelerates optical diagnostics of renal injury by 50 min compared to existing macromolecular approaches. We develop a compact molecular sensing platform for in vivo intracellular sensing, and demonstrate the versatile applications of these dyes in multispectral fluorescence and optoacoustic imaging. We find that aromaticity reversal upon electronic excitation, as indicated by magnetic descriptors, not only reduces the energy bandgap but also induces strong vibronic coupling, resulting in ultrafast excited-state dynamics and unparalleled photostability. These results support the argument for ground-state antiaromaticity as a useful design rule of dye development, enabling performances essential for modern biophotonics.

Molecular-Based FRET Nanosensor with Dynamic Ratiometric NIR-IIb Fluorescence for Real-Time In Vivo Imaging and Sensing.

Real-time fluorescence sensing can provide insight into biodynamics. However, few fluorescent tools are available to overcome the tissue scattering and autofluorescence interference for high-contrast in vivo sensing with high spatiotemporal resolution. Here, we develop a molecular-based FRET nanosensor (MFN) capable of producing a dynamic ratiometric NIR-IIb (1500-1700 nm) fluorescence signal under a frequency-modulated dual-wavelength excitation bioimaging system. The MFN provides reliable signals in highly scattering tissues and enables in vivo real-time imaging at micrometer-scale spatial resolution and millisecond-scale temporal resolution. As a proof of concept, a physiological pH-responsive nanosensor (MFNpH) was designed as a nanoreporter for intravital real-time monitoring of the endocytosis dynamics of nanoparticles in the tumor microenvironment. We also show that MFNpH allows the accurate quantification of pH changes in a solid tumor through video-rate ratiometric imaging. Our study offers a powerful approach for noninvasive imaging and sensing of biodynamics with micrometer-scale spatial resolution and millisecond-scale temporal resolution.

HClO-Activated Near-Infrared Fluorogenic Aza-BODIPY-Bisferrocene Triad with High Turn-on Ratio for In Vivo Biosensing.

Optically monitoring hypochlorous acid (HClO) in living body favors diagnosis and study of inflammatory diseases. However, this has been hampered by limited strategies to develop highly fluorogenic tools in the deep-penetration near-infrared spectrum. Herein, a near-infrared aza-BODIPY-bisferrocene triad Fc2 -CBDP that unexpectedly achieves an exceptionally sensitive and selective fluorescence turn-on (>220-fold) response toward HClO through single-ferrocene oxidation and boron-alkynyl hydrolysis cascade is reported. Mechanism insight shows that Fc2 -CBDP features "enhanced charge transfer"-caused quenching due to intramolecular bisferrocene electronic coupling, which is decoupled in the reaction with HClO. The utility of Fc2 -CBDP for intracellular HClO imaging is evaluated and, more importantly, in vivo high-contrast deep-tissue imaging of lymphatic inflammation and colitis is realized. This work provides new insights into both HClO and ferrocene chemistry, and extends the reach of fluorogenic strategies in the near-infrared biosensing.

NIR-II cell endocytosis-activated fluorescent probes for in vivo high-contrast bioimaging diagnostics.

Fluorescence probes have great potential to empower bioimaging, precision clinical diagnostics and surgery. However, current probes are limited to in vivo high-contrast diagnostics, due to the substantial background interference from tissue scattering and nonspecific activation in blood and normal tissues. Here, we developed a kind of cell endocytosis-activated fluorescence (CEAF) probe, which consists of a hydrophilic polymer unit and an acid pH-sensitive small-molecule fluorescent moiety that operates in the "tissue-transparent" second near-infrared (NIR-II) window. The CEAF probe stably presents in the form of quenched nanoaggregates in water and blood, and can be selectively activated and retained in lysosomes through cell endocytosis, driven by a synergetic mechanism of disaggregation and protonation. In vivo imaging of tumor and inflammation with a passive-targeting and affinity-tagged CEAF probe, respectively, yields highly specific signals with target-to-background ratios over 15 and prolonged observation time up to 35 hours, enabling positive implications for surgical, diagnostic and fundamental biomedical studies.

A hybrid erbium(III)-bacteriochlorin near-infrared probe for multiplexed biomedical imaging.

Spectrally distinct fluorophores are desired for multiplexed bioimaging. In particular, monitoring biological processes in living mammals needs fluorophores that operate in the 'tissue-transparent' near-infrared (NIR) window, that is, between 700 and 1,700 nm. Here we report a fluorophore system based on molecular erbium(III)-bacteriochlorin complexes with large Stokes shift (>750 nm) and narrowband NIR-to-NIR downconversion spectra (full-width at half-maximum ≤ 32 nm). We have found that the fast (2 × 109 s-¹) and near-unity energy transfer from bacteriochlorin triplets to the erbium(III) 4I13/2 level overcomes the notorious vibrational overtones quenching, resulting in bright and long-lived (1.73 µs) 1,530 nm luminescence in water. We demonstrate the excitation/emission-multiplexed capability of the complexes in the visualization of dynamic circulatory and metabolic processes in living mice, and through skull tracking of cancer cell metastases in mouse brain. This hybrid probe system facilitates robust multiplexed NIR imaging with high contrast and spatial resolution for applications ranging from fluorescence-guided surgery, diagnostics and intravital microscopy.

Surface-Confined Winding Assembly of Mesoporous Nanorods.

Bending and folding are important stereoscopic geometry parameters of one-dimensional (1D) nanomaterials, yet the precise control of them has remained a great challenge. Herein, a surface-confined winding assembly strategy is demonstrated to regulate the stereoscopic architecture of uniform 1D mesoporous SiO2 (mSiO2) nanorods. Based on this brand-new strategy, the 1D mSiO2 nanorods can wind on the surface of 3D premade nanoparticles (sphere, cube, hexagon disk, spindle, rod, etc.) and inherit their surface topological structures. Therefore, the mSiO2 nanorods with a diameter of ∼50 nm and a variable length can be bent into arc shapes with variable radii and radians, as well as folded into 60, 90, 120, and 180° angular convex corners with controllable folding times. Additionally, in contrast to conventional core@shell structures, this winding structure induces partial exposure and accessibility of the premade nanoparticles. The functional nanoparticles can exhibit large accessible surface and efficient energy exchanges with the surroundings. As a proof of concept, winding-structured CuS&mSiO2 nanocomposites are fabricated, which are made up of a 100 nm CuS nanosphere and the 1D mSiO2 nanorods with a diameter of ∼50 nm winding the nanosphere in the perimeter. The winding structured nanocomposites are demonstrated to have fourfold photoacoustic imaging intensity compared with the conventional core@shell nanostructure with an inaccessible core because of the greatly enhanced photothermal conversion efficiency (increased by ∼30%). Overall, our work paves the way to the design and synthesis of 1D nanomaterials with controllable bending and folding, as well as the formation of high-performance complex nanocomposites.

Aggressive Man-Made Red Blood Cells for Hypoxia-Resistant Photodynamic Therapy.

Extreme hypoxia of tumors represents the most notable barrier against the advance of tumor treatments. Inspired by the biological nature of red blood cells (RBCs) as the primary oxygen supplier in mammals, an aggressive man-made RBC (AmmRBC) is created to combat the hypoxia-mediated resistance of tumors to photodynamic therapy (PDT). Specifically, the complex formed between hemoglobin and enzyme-mimicking polydopamine, and polydopamine-carried photosensitizer is encapsulated inside the biovesicle that is engineered from the recombined RBC membranes. The mean corpuscular hemoglobin of AmmRBCs reaches about tenfold as high as that of natural RBCs. Owing to the same origin of outer membranes, AmmRBCs share excellent biocompatibility with parent RBCs. The introduced polydopamine plays the role of the antioxidative enzymes existing inside RBCs to effectively prevent the oxygen-carrying hemoglobin from the oxidation damage during the circulation. This biomimetic engineering can accumulate in tumors, permit in situ efficient oxygen supply, and impose strong PDT efficacy toward the extremely hypoxic tumor with complete tumor elimination. The man-made pseudo-RBC shows potentials as a universal oxygen-self-supplied platform to sensitize hypoxia-limited tumor treatment means, including but not limited to PDT. Meanwhile, this study offers ideas to the production of artificial substitutes of packed RBCs for clinical blood transfusion.