Metabolic Labeling of Peptidoglycan with NIR-II Dye Enables In Vivo Imaging of Gut Microbiota

Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real-time, and in vivo bacterial imaging method. To address this unmet need in microbial visualization, we herein report the development of a second near-infrared (NIR-II)-based method for in vivo imaging of gut bacteria. Using d -propargylglycine in gavage and then click reaction with an azide-containing NIR-II dye, gut microbiota of a donor mouse was strongly labeled with NIR-II fluorescence on their peptidoglycan. The bacteria could be readily visualized in recipient mouse gut with high spatial resolution and deep tissue penetration under NIR irradiation. The NIR-II-based metabolic labeling strategy reported herein, provides, to the best of our knowledge, the first protocol for facile in vivo visualization of gut microbiota within deep tissues, and offers an instrumental tool for deciphering the complex biology of these gut “dark matters”.     

Quantification of Bacterial Metabolic Activities in the Gut by d‐Amino Acid‐Based In Vivo Labeling

Herein, we propose a metabolic d ‐amino acid‐based labeling and in situ hybridization‐facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d ‐amino acid (FDAA)‐based labeling intensities of bacteria were found to highly correlate with their temporal and steady‐state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA‐labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram‐negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram‐positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.     

Fluorescence Imaging In Vivo at Wavelengths beyond 1500 nm

Compared to imaging in the visible and near‐infrared regions below 900 nm, imaging in the second near‐infrared window (NIR‐II, 1000–1700 nm) is a promising method for deep‐tissue high‐resolution optical imaging in vivo mainly owing to the reduced scattering of photons traversing through biological tissues. Herein, semiconducting single‐walled carbon nanotubes with large diameters were used for in vivo fluorescence imaging in the long‐wavelength NIR region (1500–1700 nm, NIR‐IIb). With this imaging agent, 3–4 μm wide capillary blood vessels at a depth of about 3 mm could be resolved. Meanwhile, the blood‐flow speeds in multiple individual vessels could be mapped simultaneously. Furthermore, NIR‐IIb tumor imaging of a live mouse was explored. NIR‐IIb imaging can be generalized to a wide range of fluorophores emitting at up to 1700 nm for high‐performance in vivo optical imaging.     

Tm3+‐Sensitized NIR‐II Fluorescent Nanocrystals for In Vivo Information Storage and Decoding

In vivo fluorescence imaging in the second near‐infrared window (NIR‐II) affords deep‐tissue penetration and high spatial resolution. Herein, we present a new type of Tm³⁺‐sensitized lanthanide nanocrystals with both excitation (1208 nm) and emission (1525 nm) located in the NIR‐II window for in vivo optical information storage and decoding. Taking advantage of the tunable fluorescence lifetimes, the optical multiplexed encoding capacity is enhanced accordingly. Micro‐devices with QR codes featuring the NIR‐II fluorescence‐lifetime multiplexed encoding were implanted into mice and were successfully decoded through time‐gated fluorescence imaging technology.     

Quantification of Bacterial Metabolic Activities in the Gut by d-Amino Acid-Based In Vivo Labeling

Herein, we propose a metabolic d -amino acid-based labeling and in situ hybridization-facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d -amino acid (FDAA)-based labeling intensities of bacteria were found to highly correlate with their temporal and steady-state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA-labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram-negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram-positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.     

NIR‐II Fluorescent Self‐Assembled Peptide Nanochain for Ultrasensitive Detection of Peritoneal Metastasis

Fluorescence‐guided cytoreductive surgery is one of the most promising approaches for facile elimination of tumors in situ, thereby improving prognosis. Reported herein is a simple strategy to construct a novel chainlike NIR‐II nanoprobe (APP‐Ag₂S‐RGD) by self‐assembly of an amphiphilic peptide (APP) into a nanochain with subsequent chemical crosslinking of NIR‐II Ag₂S QDs and the tumor‐targeting RGD peptide. This probe exhibits higher capability for cancer cell detection compared with that of RGD‐functionalized Ag₂S QDs (Ag₂S‐RGD) at the same concentration. Upon intraperitoneal injection, superior tumor‐to‐normal tissue signal ratio is achieved and non‐vascularized tiny tumor metastatic foci as small as about 0.2 mm in diameter could be facilely eliminated under NIR‐II fluorescent imaging guidance. These results clearly indicate the potential of this probe for fluorescence‐guided tumor staging, preoperative diagnosis, and intraoperative navigation.     

Real-time identification of gut microbiota with aminopeptidase N using an activable NIR fluorescent probe

A NIR fluorescent probe (DDAA) derived from fluorophore DDAO with alanine as the recognition group was developed for sensing aminopeptidase N (APN) in gut microbiota. Using DDAA as the real-time guidance tool for the fluorescence imaging of intestinal microorganism, target bacteria and saccharomycete possessing active APN were identified successfully from human feces.     

Imaging of Colorectal Cancers Using Activatable Nanoprobes with Second Near‐Infrared Window Emission

Fluorescent probes in the second near‐infrared window (NIR‐II) allow high‐resolution bioimaging with deep‐tissue penetration. However, existing NIR‐II materials often have poor signal‐to‐background ratios because of the lack of target specificity. Herein, an activatable NIR‐II nanoprobe for visualizing colorectal cancers was devised. This designed probe displays H₂S‐activated ratiometric fluorescence and light‐up NIR‐II emission at 900–1300 nm. By using this activatable and target specific probe for deep‐tissue imaging of H₂S‐rich colon cancer cells, accurate identification of colorectal tumors in animal models were performed. It is anticipated that the development of activatable NIR‐II probes will find widespread applications in biological and clinical systems.     

Organic Spherical Nucleic Acids for the Transport of a NIR‐II‐Emitting Dye Across the Blood–Brain Barrier

DNA nanotechnology plays an increasingly important role in the biomedical field; however, its application in the design of organic nanomaterials is underexplored. Herein, we report the use of DNA nanotechnology to transport a NIR‐II‐emitting nanofluorophore across the blood–brain barrier (BBB), facilitating non‐invasive imaging of brain tumors. Specifically, the DNA block copolymer, PS‐b‐DNA, is synthesized through a solid‐phase click reaction. We demonstrate that its self‐assembled structure shows exceptional cluster effects, among which BBB‐crossing is the most notable. Therefore, PS‐b‐DNA is utilized as an amphiphilic matrix to fabricate a NIR‐II nanofluorephore, which is applied in in vivo bioimaging. Accordingly, the NIR‐II fluorescence signal of the DNA‐based nanofluorophore localized at a glioblastoma is 3.8‐fold higher than the NIR‐II fluorescence signal of the PEG‐based counterpart. The notably increased imaging resolution will significantly benefit the further diagnosis and therapy of brain tumors.     

Graphene‐Oxide‐Modified Lanthanide Nanoprobes for Tumor‐Targeted Visible/NIR‐II Luminescence Imaging

The synthesis of hydrophilic lanthanide‐doped nanocrystals (Ln³⁺‐NCs) with molecular recognition ability for bioimaging currently remains a challenge. Herein, we present an effective strategy to circumvent this bottleneck by encapsulating Ln³⁺‐NCs in graphene oxide (NCs@GO). Monodisperse NCs@GO was prepared by optimizing GO size and core–shell structure of NaYF₄:Yb,Er@NaYF₄, thus combining the intense visible/near‐infrared II (NIR‐II) luminescence of NCs and the unique surface properties and biomedical functions of GO. Such nanostructures not only feature broad solvent dispersibility, efficient cell uptake, and excellent biocompatibility but also enable further modifications with various agents such as DNA, proteins, or nanoparticles without tedious procedures. Moreover, we demonstrate in proof‐of‐concept experiments that NCs@GO can realize simultaneous intracellular tracking and microRNA‐21 visualization, as well as highly sensitive in vivo tumor‐targeted NIR‐II imaging at 1525 nm.     

Nanoaggregate Probe for Breast Cancer Metastasis through Multispectral Optoacoustic Tomography and Aggregation‐Induced NIR‐I/II Fluorescence Imaging

An activatable nanoprobe for imaging breast cancer metastases through near infrared‐I (NIR‐I)/NIR‐II fluorescence imaging and multispectral optoacoustic tomography (MSOT) imaging was designed. With a dihydroxanthene moiety serving as the electron donor, quinolinium as the electron acceptor and nitrobenzyloxydiphenylamino as the recognition element, the probe can specifically respond to nitroreductase and transform into an activated D‐π‐A structure with a NIR emission band extending beyond 900 nm. The activated nanoprobe exhibits NIR emission enhanced by aggregation‐induced emission (AIE) and produces strong optoacoustic signal. The nanoprobe was used to detect and image metastases from the orthotopic breast tumors to lymph nodes and then to lung in two breast cancer mouse models. Moreover, the nanoprobe can monitor the treatment efficacy during chemotherapeutic course through fluorescence and MSOT imaging.     

Naked‐Eye Readout of Analyte‐Induced NIR Fluorescence Responses by an Initiation–Input–Transduction Nanoplatform

Fluorescence visualization (FV) in the near‐infrared (NIR) window promises to break through the signal‐to‐background ratio (SBR) bottleneck of traditional visible‐light‐driven FV methods. However, straightforward NIR‐FV has not been realized, owing to the lack of methods to readily transduce NIR responses into instrument‐free, naked eye‐recognizable outputs. Now, an initiation–input–transduction platform comprising a well‐designed NIR fluorophore as the signal initiator and lanthanide‐doped nanocrystals as the transducer for facile NIR‐FV is presented. The analyte‐induced off–on NIR signal serves as a sensitizing switch of transducer visible luminescence for naked‐eye readout. The design is demonstrated for portable, quantitative detection of phosgene with significantly improved SBR and sensitivity. By further exploration of initiators, this strategy holds promise to create advanced NIR‐FV probes for broad sensing applications.     

High‐Performance Quinoline‐Malononitrile Core as a Building Block for the Diversity‐Oriented Synthesis of AIEgens

In vivo fluorescent monitoring of physiological processes with high‐fidelity is essential in disease diagnosis and biological research, but faces extreme challenges due to aggregation‐caused quenching (ACQ) and short‐wavelength fluorescence. The development of high‐performance and long‐wavelength aggregation‐induced emission (AIE) fluorophores is in high demand for precise optical bioimaging. The chromophore quinoline‐malononitrile (QM) has recently emerged as a new class of AIE building block that possesses several notable features, such as red to near‐infrared (NIR) emission, high brightness, marked photostability, and good biocompatibility. In this minireview, we summarize some recent advances of our established AIE building block of QM, focusing on the AIE mechanism, regulation of emission wavelength and morphology, the facile scale‐up and fast preparation for AIE nanoparticles, as well as potential biomedical imaging applications.     

Antibiotics-based fluorescent probes for selective labeling of Gram-negative and Gram-positive bacteria in living microbiotas

Gram-staining distinguishes bacteria into two major groups,Gram-positive and Gram-negative bacteria,and has become an essential technique in microbiology.However,Gram-staining is not compatible with living cells and thus limits its applications.Here,we report the development of a polymyxin B-based fluorescent probe that enables selective labeling of Gram-negative in the living microbiota samples.We first synthesized the polymyxin B-Cy3 conjugate and confirmed its specificity for labeling Gram-negative bacteria.In combination with a previously developed Gram-positive-specific fluorescent probe,we demonstrate two-color imaging of Gram-positive and Gram-negative bacteria in various kinds living microbiotas,including mouse gut,human oral,soil,and crude oil microbiotas,with high selectivity and coverage.Finally,a pilot use of the probes in staining bacteria on heat-fixed sputum smear was also demonstrated,showing its potentials in clinical microbiology.Our method provides a versatile tool for distinguishing Gram-positive and Gram-negative bacteria in both basic research and clinical settings.     

Stable,Wavelength‐Tunable Fluorescent Dyes in the NIR‐II Region for In Vivo High‐Contrast Bioimaging and Multiplexed Biosensing

Small‐molecule organic fluorophores, spectrally active in the 900–1700 nm region, with tunable wavelength and sensing properties are sought‐after for in vivo optical imaging and biosensing. A panel of fluorescent dyes ( CX ) has been developed to meet this challenge. CX dyes exhibit the wavelength tunability of cyanine dyes and have a rigidified polymethine chain to guarantee their stability. They are chemo‐ and photo‐stable in an aqueous environment and have tunable optical properties with maximal absorbing/emitting wavelength at 1089/1140 nm. They show great potential in high‐contrast in vivo bioimaging and multicolor detection with negligible optical cross talk. Förster resonance energy transfer (FRET) between CX dyes was demonstrated in deep tissue, providing an approach for monitoring drug‐induced hepatotoxicity by detection of OONO^?. This report presents a series of NIR‐II dyes with promising spectroscopic properties for high‐contrast bioimaging and multiplexed biosensing.     

Synthesis of a Near‐Infrared BODIPY Dye for Bioimaging and Photothermal Therapy

The development of robust photothermal agents for near‐infrared (NIR) imaging is a great challenge. Herein, we report the design and synthesis of a new photothermal agent, based on the aza‐boron‐dipyrromethene framework (azaBDP). This compound possessed excellent photostability and high photothermal‐conversion efficiency (50 %) under NIR laser irradiation. When the photothermal properties of this compound were utilized for tumor inhibition, stable long‐term fluorescence was observed in living animals. Photothermal treatment efficiently suppressed tumor growth, as evidenced by in vitro and in vivo experiments. Furthermore, NIR emission could be detected by using an imaging system and therapeutic self‐monitoring was achieved by using NIR imaging.     

Stable,Wavelength‐Tunable Fluorescent Dyes in the NIR‐II Region for In Vivo High‐Contrast Bioimaging and Multiplexed Biosensing

Small‐molecule organic fluorophores, spectrally active in the 900–1700 nm region, with tunable wavelength and sensing properties are sought‐after for in vivo optical imaging and biosensing. A panel of fluorescent dyes ( CX ) has been developed to meet this challenge. CX dyes exhibit the wavelength tunability of cyanine dyes and have a rigidified polymethine chain to guarantee their stability. They are chemo‐ and photo‐stable in an aqueous environment and have tunable optical properties with maximal absorbing/emitting wavelength at 1089/1140 nm. They show great potential in high‐contrast in vivo bioimaging and multicolor detection with negligible optical cross talk. Förster resonance energy transfer (FRET) between CX dyes was demonstrated in deep tissue, providing an approach for monitoring drug‐induced hepatotoxicity by detection of OONO^?. This report presents a series of NIR‐II dyes with promising spectroscopic properties for high‐contrast bioimaging and multiplexed biosensing.     

Zinc(II)‐Dipicolylamine Coordination Nanotheranostics: Toward Synergistic Nanomedicine by Combined Photo/Gene Therapy

We report the rational design of coordination‐driven self‐assembly metal–organic nanostructures for multifunctional nanotheranostics. Zinc(II) coordination‐based nano‐formulations capable of loading indocyanine green (ICG) and therapeutic genes were prepared to achieve a fluorescence/photoacoustic imaging‐guided combination photo/gene therapy strategy. We showed the enhanced theranostic capability of zinc(II)‐dipicolylamine‐assisted assembly of ICG, as well as simultaneous targeted gene delivery in an experimental mouse model of cancer. Such a co‐assembly strategy provides a facile way to achieve combined therapeutic functions for personalized nanomedicine.     

Mo2C‐Derived Polyoxometalate for NIR‐II Photoacoustic Imaging‐Guided Chemodynamic/Photothermal Synergistic Therapy

To overcome the current limitations of chemodynamic therapy (CDT), a Mo₂C‐derived polyoxometalate (POM) is readily synthesized as a new CDT agent. It permits synergistic chemodynamic and photothermal therapy operating in the second near‐infrared (NIR‐II) biological transparent window for deep tissue penetration. POM aggregated in an acidic tumor micro‐environment (TME) whereby enables specific tumor targeting. In addition to the strong ability to produce singlet oxygen (¹O₂) presumably via Russell mechanism, its excellent photothermal conversion enhances the CDT effect, offers additional tumor ablation modality, and permits NIR‐II photoacoustic imaging. Benefitting from the reversible redox property of molybdenum, the theranostics based on POM can escape from the antioxidant defense system. Moreover, combining the specific responsiveness to TME and localized laser irradiation, side‐effects shall be largely avoided.     

Doubly Strapped Zwitterionic NIR-I and NIR-II Heptamethine Cyanine Dyes for Bioconjugation and Fluorescence Imaging

Heptamethine cyanine dyes enable deep tissue fluorescence imaging in the near infrared (NIR) window. Small molecule conjugates of the benchmark dye ZW800-1 have been tested in humans. However, long-term imaging protocols using ZW800-1 conjugates are limited by their instability, primarily because the chemically labile C4′-O-aryl linker is susceptible to cleavage by biological nucleophiles. Here, we report a modular synthetic method that produces novel doubly strapped zwitterionic heptamethine cyanine dyes, including a structural analogue of ZW800-1 , with greatly enhanced dye stability. NIR-I and NIR-II versions of these doubly strapped dyes can be conjugated to proteins, including monoclonal antibodies, without causing undesired fluorophore degradation or dye stacking on the protein surface. The fluorescent antibody conjugates show excellent tumor-targeting specificity in a xenograft mouse tumor model. The enhanced stability provided by doubly strapped molecular design will enable new classes of in vivo NIR fluorescence imaging experiments with possible translation to humans.