Fluorescence Imaging Research Articles

Novel Far-Red Fluorescent 1,4-Dihydropyridines for L-Type Calcium Channel Imaging.

Upregulation of L-type calcium channels (LTCCs) is implicated in a range of cardiovascular and neurological disorders. Therefore, the development of toolboxes that unlock fast imaging protocols in live cells is coveted. Herein, we report a library of first-in-class far-red small-molecule-based fluorescent ligands (FluoDiPines), able to target LTCCs. All fluorescent ligands were evaluated in whole-cell patch-clamp and live-cell Ca2+ imaging whereby FluoDiPine 6 was found to be the best candidate for live-cell fluorescence imaging. Low concentration of FluoDiPine 6 (50 nM) and a quick labeling protocol (5 min) are successfully applied to fixed and live cells to image LTCCs with good specificity.

Exploring Transient States of PAmKate to Enable Improved Cryogenic Single-Molecule Imaging.

Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nanoscale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) that photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remain a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find that a transient state forms with a lifetime of ∼2 ms under cryogenic conditions, which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for the study of other FPs at cryogenic temperatures.

Lymphatic magnetic resonance imaging abnormalities in children with repaired tetralogy of Fallot.

Tetralogy of Fallot patients face an elevated risk of developing chylothorax and pleural effusions post-surgery. This patient group exhibits risk factors known to compromise the lymphatic system, such as elevated central venous pressure, pulmonary flow changes, and hypoxia. This study investigates the morphology and function of the lymphatic system in tetralogy of Fallot patients through lymphatic magnetic resonance imaging and near-infrared fluorescence imaging, respectively. Post-repair tetralogy of Fallot patients aged 6-18 years were recruited, along with age and gender-matched controls. Magnetic resonance imaging was used to assess the morphology of the thoracic lymphatic vessels and the thoracic, while near-infrared fluorescence imaging was used to assess lymphatic activity utilising lymph rate, velocity, and pressure. Nine patients and 10 controls were included. Echocardiography revealed that 2/3 of the patients had moderate-severe pulmonary regurgitation, while none displayed signs of elevated central venous pressure. Magnetic resonance imaging identified three patients with type 3 (out of 4 types) lymphatic abnormalities, while controls had none. The thoracic ducts showed severe (one patient) and moderate (one patient) tortuosity. Mean thoracic duct diameters were 3.3 mm ±1.1 in patients and 3.0 mm ± 0.8 in controls (p-value = 0.53). Near-infrared fluorescence imaging revealed no anomalous patterns. Despite no presence of clinical lymphatic disease, 3/9 of the repaired tetralogy of Fallot patients exhibited lymphatic morphological abnormalities. The significance of these anomalies remains uncertain currently. Further research is needed to determine whether these lymphatic alterations in this patient cohort are a result of congenital malformations, haemodynamic shifts, or prenatal and early-life saturation levels.

NanoPlex: a universal strategy for fluorescence microscopy multiplexing using nanobodies with erasable signals

Fluorescence microscopy has long been a transformative technique in biological sciences. Nevertheless, most implementations are limited to a few targets, which have been revealed using primary antibodies and fluorescently conjugated secondary antibodies. Super-resolution techniques such as Exchange-PAINT and, more recently, SUM-PAINT have increased multiplexing capabilities, but they require specialized equipment, software, and knowledge. To enable multiplexing for any imaging technique in any laboratory, we developed NanoPlex, a streamlined method based on conventional antibodies revealed by engineered secondary nanobodies that allow the selective removal of fluorescence signals. We develop three complementary signal removal strategies: OptoPlex (light-induced), EnzyPlex (enzymatic), and ChemiPlex (chemical). We showcase NanoPlex reaching 21 targets for 3D confocal analyses and 5–8 targets for dSTORM and STED super-resolution imaging. NanoPlex has the potential to revolutionize multi-target fluorescent imaging methods, potentially redefining the multiplexing capabilities of antibody-based assays.

Strongly quenched activatable theranostic nanogel for precision imaging-guided photodynamic therapy and enhanced immunotherapy

Immune checkpoint inhibitors (ICIs) are innovative immunotherapeutic agents for cancer. However, their low therapeutic efficacy in patients with large or rapidly growing tumors, along with their high cost, represents a notable limitation in their clinical applications. Therefore, new and safe strategies must be developed to enhance the therapeutic efficacy of ICIs in clinical settings. In this study, we developed a near-infrared (NIR) fluorescent dye-loaded activatable theranostic nanogel (NATNgel) for precision imaging-guided photodynamic therapy (PDT) and combined immunotherapy for rapidly growing tumors. Although NIR fluorescence and phototoxicity of NATNgel are strongly quenched, these can be selectively activated inside target tumor cells. A high tumor-to-background ratio (7.31 ± 1.40) in NIR fluorescence imaging could be achieved in NATNgel-treated mice, enabling real-time image-guided PDT. The combination of PDT and anti-PD-1 antibody therapy resulted in complete tumor regression. Histopathological evaluation of major organs and blood chemistry analysis revealed no side effects of the combined treatment regimen. In addition, the combination treatment completely suppressed the growth of rechallenged tumors. Overall, NATNgel is a safe and promising theranostic material for precision imaging-guided PDT and enhanced immunotherapy.

A pH-responsive fluorescent film with the smartphone-assistance for real-time and visual detection of food freshness

Monitoring food freshness is considerably important for food safety. In this study, a smart pH-responsive fluorescence hydroxypropyl methyl cellulose-κ-carrageenan-fluorescein isothiocyanate-NH2-CaAl2O4 (H-K-F-N) film was prepared. Taking synergetic advantage of the pH-dependent behavior of fluorescein isothiocyanate dye and the luminescence characteristics of calcium aluminate phosphor, the film exhibited a unique strong pH-responsive fluorescence with an exceptional linear relationship (correlation coefficient, R2 = 0.9993) across a wide pH rang of 2.0–12.0. Moreover, the H-K-F-N film, as a smart sensor, could be used to estimate the total volatile basic nitrogen and total viable count through fluorescence intensity based on the partial least squares regression model and support vector machine regression, respectively. Leveraging the relationship between the fluorescent image's digital signals and food freshness indicators, a smartphone-assisted system was developed. These results demonstrated that H-K-F-N film is promising for applications in intelligent food packaging and food safety monitoring.

Assessing the Tumor Immune Landscape Across Multiple Spatial Scales to Differentiate Immunotherapy Response in Metastatic Non-Small Cell Lung Cancer

Although immune checkpoint inhibitor-based therapy has shown promising results in non-small cell lung cancer patients with high programmed death-ligand 1 expression, not all patients respond to therapy. The tumor microenvironment (TME) is complex and heterogeneous, making it challenging to understand the key agents and features that influence response to therapies. In this study, we leverage multiplex fluorescent immunohistochemistry to quantitatively assess interactions between tumor and immune cells in an effort to identify patterns occurring at multiple spatial levels of the TME. To do so, we introduce several computational methods novel to a data set of 1,269 multiplex fluorescent immunohistochemistry images from a cohort of 52 patients with metastatic non-small cell lung cancer. With the spatial G-cross function, we quantify the degree of cell interaction at an entire image level, where we see significantly increased activity of cytotoxic T cells and helper T cells with epithelial tumor cells in responders to immune checkpoint inhibitor-based (P = .022 and P < .001, respectively) and decreased activity of T-regulatory cells with epithelial tumor cells compared with nonresponders (P = .010). By leveraging spatial overlap methods, we define tumor subregions (which we call the tumor “periphery,” “edge.” and “center”) and discover more localized immune-immune interactions influencing positive response, including those between cytotoxic T cells and helper T cells with antigen presenting cells in these subregions specifically. Finally, we trained an interpretable deep learning model that identified key cellular regions of interest that most influenced response classification (area under the curve = 0.71 ± 0.02). Assessing spatial interactions within these subregions further revealed new insights that were not significant at the whole image level, particularly the elevated association of antigen presenting cells and T-regulatory cells with one another in responder groups (P = .024). Altogether, we demonstrate that elucidating patterns of cell composition and interplay across multiple levels of spatial analyses can improve our understanding of the TME and better differentiate patient responses to immunotherapy.

Novel anti-CEA affibody for rapid tumor-targeting and molecular imaging diagnosis in mice bearing gastrointestinal cancer cell lines.

Gastrointestinal cancer is a common malignant tumor with a high incidence worldwide. Despite continuous improvements in diagnosis and treatment strategies, the overall prognosis of gastrointestinal tumors remains poor. Carcinoembryonic antigen (CEA) is highly expressed in various types of cancers, especially in gastrointestinal cancers, making it a potential target for therapeutic intervention. Therefore, the expression of CEA can be used as an indication of the existence of tumors, chosen as a target for molecular imaging diagnosis, and effectively utilized in the targeted therapy of gastrointestinal cancers. In this study, we report the selection and characterization of affibody molecules (ZCEA539, ZCEA546, and ZCEA919) specific to the CEA protein. Their ability to bind to recombinant and native CEA protein has been confirmed by surface plasmon resonance (SPR), immunofluorescence, and immunohistochemistry assays. Furthermore, Dylight755-labeled ZCEA affibody showed accumulation within the tumor site 1 h post injection and was continuously enhanced for 4 h. The Dylight755-labeled ZCEA affibody exhibited high tumor-targeting specificity in CEA+ xenograft-bearing mice and possesses promising characteristics for tumor-targeting imaging. Overall, our results suggest the potential use of ZCEA affibodies as fluorescent molecular imaging probes for detecting CEA expression in gastrointestinal cancer.

Highly fluorescent nitrogen doped carbon dots as analytical probe for sensitive detection of curcumin through smartphone integrated 3D-printed platform: A new horizon in food safety

COVID-19 pandemic has significantly influenced the dietary habits of humans, emphasizing the incorporation of natural ingredients to enhance immunity towards viral and bacterial infections. Curcumin (Cur), a widely used traditional medicine in various Asian countries and a natural coloring agent, has gained popularity, leading to surge in its usage specially in post COVID-19 era. This surge has led to increased scrutiny of the potential side effects of excessive Cur use, with recent reports suggesting it may result in inactivation of DNA and reduce adenosine triphosphate levels, leading to health risks. In this work, we synthesized highly fluorescent nitrogen-doped carbon dots with a photoluminescence quantum yield of 72.9 % for the sensitive and selective detection of Cur. The developed fluorescent probe exhibits excellent sensory response towards Cur within a concentration range of 0.081–51.45 µM, achieving an ultra-low detection limit of 15.91 nM. The sensor was successfully tested on real food samples like ginger powder, turmeric powder, and curry powder, demonstrating good recovery rates. To assess the practicality of the sensor system, we developed a 3D-printed smartphone-integrated device platform for curcumin detection through fluorescence image analysis. This developed platform exhibited promising results, achieving a limit of detection (LoD) of 132.28 nM across a curcumin concentration range of 0.13–54.00 µM. This device platform holds significant potential for the development of efficient sensors for real-time detection of Cur in food samples.

Deep learning pipeline for automated cell profiling from cyclic imaging

Cyclic fluorescence microscopy enables multiple targets to be detected simultaneously. This, in turn, has deepened our understanding of tissue composition, cell-to-cell interactions, and cell signaling. Unfortunately, analysis of these datasets can be time-prohibitive due to the sheer volume of data. In this paper, we present CycloNET, a computational pipeline tailored for analyzing raw fluorescent images obtained through cyclic immunofluorescence. The automated pipeline pre-processes raw image files, quickly corrects for translation errors between imaging cycles, and leverages a pre-trained neural network to segment individual cells and generate single-cell molecular profiles. We applied CycloNET to a dataset of 22 human samples from head and neck squamous cell carcinoma patients and trained a neural network to segment immune cells. CycloNET efficiently processed a large-scale dataset (17 fields of view per cycle and 13 staining cycles per specimen) in 10 min, delivering insights at the single-cell resolution and facilitating the identification of rare immune cell clusters. We expect that this rapid pipeline will serve as a powerful tool to understand complex biological systems at the cellular level, with the potential to facilitate breakthroughs in areas such as developmental biology, disease pathology, and personalized medicine.

Real-time robust liver and gallbladder segmentation during laparoscopic cholecystectomy using convolutional neural networks: an analysis

Aim: Images in different laparoscopic cholecystectomy datasets are acquired using various camera models, parameters, and settings, with the annotation methods varying by institution. These factors result in inconsistent inference performance of the network model. This study aims to identify the optimal network model architecture for liver and gallbladder segmentation from several options. Then, the performance and robustness of the optimal network model are evaluated using an independent dataset that is not included in the training. Methods: The public dataset, CholecSeg8k, was utilized as the input for the network model training, validation, and testing. A local private dataset from KPJ Damansara Hospital, Selangor, Malaysia, was used for testing purposes only. For the implementation of liver and gallbladder segmentation, segmentation models, a public Python library was employed. Results: Among the experiments, highly accurate liver and gallbladder segmentation results were achieved using the feature pyramid network (FPN) architecture as the network model, with the Inception-ResNet-v2 architecture as the network backbone. The best-trained network model resulted in a loss of 0.070955, a mean intersection over union (IoU) score of 0.95896, and a mean F1-score of 0.9773 on the test set. However, visualized results for the private dataset contained considerable false-negative areas. Conclusion: The proposed automated technique has the potential to serve as an alternative to the conventional indocyanine green injection along with near-infrared fluorescence imaging (ICG-NIRF)-based method for liver and gallbladder segmentation during laparoscopic cholecystectomy. Future work will focus on enhancing the results of the private dataset. Additionally, a surgeon-assistant robotic arm that will use the liver and gallbladder segmentation results for camera steering will be analyzed.

Organic probes for three‐photon fluorescence imaging in NIR‐II window: Design, applications, and perspectives

AbstractThree‐photon fluorescence (3PF) imaging is an emerging technology for imaging deep‐tissue submicroscopic structures by nonlinearly redshifting the excitation wavelength to the second near‐infrared (NIR‐II) window; thus, this approach has great advantages, including deep penetration depth, good spatial resolution, low background, and a high signal‐to‐noise ratio. 3PF imaging has been demonstrated to be a powerful tool for noninvasively visualizing all kinds of deep tissues in recent years. Benefiting from excellent biosecurity and structural controllability, the development of organic 3PF probes is highly important for advancing 3PF imaging in vivo. However, there is no summary of the generalizability of the design and recent progress in organic 3PF probes. Herein, this review introduces the fundamental principle of 3PF imaging and highlights the advantages of 3PF bioimaging. The molecular design of these organic 3PF probes is also summarized based on relative optical indices. Furthermore, different 3PF imaging application scenarios are listed in detail. In the end, the main challenges, significance of probe exploitation, and prospective orientation of organic probes for precise 3PF imaging are proposed and discussed for promoting future applications and clinical translation.

A visible light-activated azo-fluorescent switch for imaging-guided and light-controlled release of antimycotics

Azo switches are widely employed as essential components in light-responsive systems. Here, we develop an azo-fluorescent switch that is visible light-responsive and its light-responsive processes can be monitored using fluorescence imaging. Visible light irradiation promotes isomerization, accompanied by changes in fluorescence that enable the process to be monitored through fluorescence imaging. Furthermore, we document that the nanocavity size of liposome encapsulated nanoparticles containing azo changes in the isomerization process and show that this change enables construction of a light-responsive nanoplatform for optically controlled release of antimycotics. Also, natural light activation of nanoparticles of the switch loaded with an antimycotic agent causes death of Rhizoctonia solani. The results show that these nanoparticles can double the holding period in comparison to small molecule antimycotics. The strategy used to design the imaging-guided light-controlled nano-antimycotic release system can be applicable to protocols for controlled delivery of a wide variety of drugs.

Synthesis of a novel water-soluble pyridine dicarboxylate and its application in fluorescence cell imaging

Abstract A novel water-soluble substituted pyridine dicarboxylate containing a quaternary ammonium ion with potential applications in fluorescence cell imaging was synthesized and characterized. Remarkably, this compound exhibited water solubility in the absence of additional solubilizers, enabling direct dissolution in phosphate-buffered saline for cell studies. No obvious cytotoxicity was observed in 4T1 cells (mouse breast cancer cells) within the concentration range of 0.2–25 μmol L−1, with a cell survival rate above 89%. Furthermore, the compound exhibited a maximum two-photon absorption cross-section of 86 GM at an excitation wavelength of 780 nm, demonstrating high cell permeability, effective distribution in the cytoplasm, and preferential targeting of organelles. Single- and two-photon fluorescence imaging of cells revealed a distinctive blue emission and strong bright green emission, respectively. The newly synthesized substituted pyridine dicarboxylate exhibited low cytotoxicity and strong fluorescence imaging properties, demonstrating its potential in cell imaging applications.

Improving the accuracy of SIF quantified from moderate spectral resolution airborne hyperspectral imager using SCOPE: assessment with sub-nanometer imagery

Hyperspectral imaging of solar-induced chlorophyll fluorescence (SIF) is required for plant phenotyping and stress detection. However, the most accurate instruments for SIF quantification, such as sub-nanometer (≤1-nm full-width at half-maximum, FWHM) airborne hyperspectral imagers, are expensive and uncommon. Previous studies have demonstrated that standard narrow-band hyperspectral imagers (i.e., 4–6-nm FWHM) are more cost-effective and can provide far-red SIF quantified at 760 nm (SIF760), which correlates strongly with precise sub-nanometer resolution measurements. Nevertheless, narrow-band SIF760 quantifications are subject to systematic overestimation owing to the influence of the spectral resolution (SR). In this study, we propose a modelling approach based on the Soil Canopy Observation, Photochemistry and Energy Fluxes (SCOPE) model with the objective of enhancing the accuracy of absolute SIF760 levels derived from standard airborne hyperspectral imagers in practical settings. The performance of the proposed method was evaluated using airborne imagery acquired from two airborne hyperspectral imagers (FWHM ≤ 0.2-nm and 5.8-nm) flown in tandem on board an aircraft that collected data from two different wheat and maize phenotyping trials. Leaf biophysical and biochemical traits were first estimated from airborne narrow-band reflectance imagery and subsequently used as SCOPE model inputs to simulate a range of top-of-canopy (TOC) radiance and SIF spectra at 1-nm FWHM. The SCOPE simulated radiance spectra were then convolved to match the spectral configuration of the narrow-band imager to compute the 5.8-nm FWHM SIF760. A site-specific model was constructed by employing the convolved 5.8-nm SR SIF760 as the independent variable and the 1-nm SR SIF760 directly simulated by SCOPE as the dependent variable. When applied to the airborne dataset, the estimated SIF760 at 1-nm SR from the standard narrow-band hyperspectral imager matched the reference sub-nanometer quantified SIF760 with root mean square error (RMSE) less than 0.5 mW/m2/nm/sr, yielding R2 = 0.93–0.95 from the two experiments. These results suggest that the proposed modelling approach enables the interpretation of SIF760 quantified using standard hyperspectral imagers of 4–6 nm FWHM for stress detection and plant physiological condition assessment.

Development of a Fluorescent Probe with High Selectivity based on Thiol-ene Click Nucleophilic Cascade Reactions for Delving into the Action Mechanism of Serotonin in Depression.

The intrinsic correlation between depression and serotonin (5-HT) is a highly debated topic, with significant implications for the diagnosis, treatment, and advancement of drugs targeting neurological disorders. To address this important question, it is of utmost priority to understand the action mechanism of serotonin in depression through fluorescence imaging studies. However, the development of efficient molecular probes for serotonin is hindered by the lack of responsive sites with high selectivity for serotonin at the present time. Herein, we developed the first highly selective serotonin responsive site, 3-mercaptopropionate, utilizing thiol-ene click cascade nucleophilic reactions. The novel responsive site was then employed to construct the powerful molecular probe SJ-5-HT for imaging the serotonin level changes in the depression cells and brain tissues. Importantly, the imaging studies reveal that the level of serotonin in patients with depression may not be the primary factor, while the ability of neurons in patients with depression to release serotonin appears to be more critical. Additionally, this serotonin release capability correlates strongly with the levels of mTOR (intracellular mammalian target of rapamycin). These discoveries could offer valuable insights into the molecular mechanisms underpinning depression and furnish mTOR as a novel direction for the advancement of antidepressant therapies.

Luminescence Modulation in Boron-Cluster-Based Luminogens via Boron Isotope Effects.

Recent advances in luminescent materials highlight the significant impact of hydrogen isotope effects on improving optoelectronic properties. However, the research on the influence of the boron isotope effects on photophysical properties remains underdeveloped. This study focused on exploring the boron isotope effects in boron-cluster-based luminogens. In doing so, we designed and synthesized carborane-based luminogens containing 98% 10B and 95% 11B, respectively, and observed distinct photophysical behaviors. Compared to the 10B-enriched luminogens, the 11B-enriched counterparts can significantly enhance luminescence efficiency, prolong emission lifetime, and reduce full-width at half-maximum. Additionally, increased thermal stability, redshifted B-H vibrations, and a fourfold enhanced electrochemiluminescence intensity have also been observed. On the other hand, the biological assessments of a 10B-enriched luminogen reveals low cytotoxicity, high boron uptake, and excellent fluorescence imaging capability, indicating the potential application in boron neutron capture therapy (BNCT). This work presents the first comprehensive exploration on the boron isotope effects in boron clusters, and provides valuable insights into the rational design of organic luminogens for advanced optoelectronic and biomedical applications.

Luminescence Modulation in Boron‐Cluster‐Based Luminogens via Boron Isotope Effects

Recent advances in luminescent materials highlight the significant impact of hydrogen isotope effects on improving optoelectronic properties. However, the research on the influence of the boron isotope effects on photophysical properties remains underdeveloped. This study focused on exploring the boron isotope effects in boron‐cluster‐based luminogens. In doing so, we designed and synthesized carborane‐based luminogens containing 98% 10B and 95% 11B, respectively, and observed distinct photophysical behaviors. Compared to the 10B‐enriched luminogens, the 11B‐enriched counterparts can significantly enhance luminescence efficiency, prolong emission lifetime, and reduce full‐width at half‐maximum. Additionally, increased thermal stability, redshifted B–H vibrations, and a fourfold enhanced electrochemiluminescence intensity have also been observed. On the other hand, the biological assessments of a 10B‐enriched luminogen reveals low cytotoxicity, high boron uptake, and excellent fluorescence imaging capability, indicating the potential application in boron neutron capture therapy (BNCT). This work presents the first comprehensive exploration on the boron isotope effects in boron clusters, and provides valuable insights into the rational design of organic luminogens for advanced optoelectronic and biomedical applications.

Immunohistochemical Evaluation of Potential Biomarkers for Targeted Intraoperative Fluorescence Imaging in Endometriosis: Towards Optimizing Surgical Treatment.

Surgical intervention for endometriosis is an important treatment modality, yet incomplete resection resulting from poor visibility of affected tissue and consequently recurrence of disease remains a prevalent challenge. Intra-operative visualization of endometriosis, enabling fluorescence-guided surgery (FGS), could help to optimize surgical treatment. A biomarker, upregulated in endometriosis compared to adjacent tissue, is required to use as a target for FGS. Immunohistochemistry was used to evaluate protein expression of a selection of previously identified potential biomarkers. Ten biomarkers were stained in a large cohort of 84 tissues, both deep and peritoneal endometriosis and tissue without endometriosis, all from patients with confirmed endometriosis. MMP11 and VCAN showed the largest upregulation in endometriosis compared to adjacent tissue and showed a membranous or extracellular staining pattern. MMP11 is a promising target for glandular and stromal visualization, VCAN for stromal visualization only. For both biomarkers, upregulation was high in both peritoneal and deep endometriosis and for patients with and without hormonal medication. Other stained biomarkers showed non-beneficial characteristics based on staining pattern or upregulation. Analysis of all endometriosis samples showed that combined glandular and stromal targeting is expected to result in optimal visualization of endometriosis. Further research is needed to determine whether targeting one biomarker is sufficient for this goal, or if dual targeting is necessary. Development of clinical tracers for VCAN and MMP11 is necessary.

Microenvironment-Activatable Probe for Precise NIR-II Monitoring and Synergistic Immunotherapy in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) represents an insidious autoimmune inflammatory disorder that severely lowers the life quality by progressively destructing joint functions and eventually causing permanent disability, posing a serious public health problem. Here, an advanced theranostic probe is introduced that integrates activatable second near-infrared (NIR-II) fluorescence imaging for precise RA diagnosis with multi-pronged RA treatments. A novel molecular probe comprising a long-wavelength aggregation-induced emission unit and a manganese carbonyl cage motif is synthesized, which enables NIR-II fluorescence activation and concurrently releasing therapeutic carbon monoxide (CO) gas in inflamed joint microenvironment. This molecular probe self-assembles into a biocompatible nanoprobe, which is subsequently conjugated with anti-IL-6R antibody to afford active-targeting ability of RA. The nanoprobe exhibits significant turn-on NIR-II fluorescence signal at the RA lesion, enabling highly sensitive RA diagnosis and real-time therapeutic monitoring. The combination of ROS scavenging, on-demand CO gas release, and IL-6 signaling blockade results in potent therapeutic effect and synergistic immunomodulation impact, significantly alleviating the RA symptoms and preventing joint destruction. This research introduces a novel paradigm for the development of high-performance, activatable theranostic strategies to facilitate precise detection and enhanced treatment of RA-related diseases.