Hierarchical Self-Assembly of Multidimensional Functional Materials from Sequence-Defined Peptoids.

Hierarchical self-assembly represents a powerful strategy for the fabrication of functional materials across various length scales. However, achieving precise formation of functional hierarchical assemblies remains a significant challenge and requires a profound understanding of molecular assembly interactions. In this study, we present a molecular-level understanding of the hierarchical assembly of sequence-defined peptoids into multidimensional functional materials, including twisted nanotube bundles serving as a highly efficient artificial light harvesting system. By employing synchrotron-based powder X-ray diffraction and analyzing single crystal structures of model compounds, we elucidated the molecular packing and mechanisms underlying the assembly of peptoids into multidimensional nanostructures. Our findings demonstrate that incorporating aromatic functional groups, such as tetraphenyl ethylene (TPE), at the termini of assembling peptoid sequences promotes the formation of twisted bundles of nanotubes and nanosheets, thus enabling the creation of a highly efficient artificial light harvesting system. This research exemplifies the potential of leveraging sequence-defined synthetic polymers to translate microscopic molecular structures into macroscopic assemblies. It holds promise for the development of functional materials with precisely controlled hierarchical structures and designed functions.

Dynamic nitrogen fixation in an aerobic endophyte of Populus.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation.

Multiplexed molecular profiling of tissue microenvironments, or spatial omics, can provide critical insights into cellular functions and disease pathology. The coupling of laser microdissection with mass spectrometry-based proteomics has enabled deep and unbiased mapping of >1000 proteins. However, the throughput of laser microdissection is often limited due to tedious two-step procedures, sequential laser cutting, and sample collection. The two-step procedure also hinders the further improvement of spatial resolution to <10 µm as needed for subcellular proteomics. Herein, we developed a high-throughput and high-resolution spatial proteomics platform by seamlessly coupling deep ultraviolet (DUV) laser ablation (LA) with nanoPOTS (Nanodroplet Processing in One pot for Trace Samples)-based sample preparation. We demonstrated the DUV-LA system can quickly isolate and collect tissue samples at a throughput of ∼30 spots/min and a spatial resolution down to 2 µm from a 10 µm thick human pancreas tissue section. To improve sample recovery, we developed a proximity aerosol collection approach by placing DMSO droplets close to LA spots. We demonstrated the DUV-LA-nanoPOTS platform can detect an average of 1312, 1533, and 1966 proteins from ablation spots with diameters of 7, 13, and 19 µm, respectively. In a proof-of-concept study, we isolated and profiled two distinct subcellular regions of the pancreas tissue revealed by hematoxylin and eosin (H&E) staining. Quantitative proteomics revealed proteins specifically enriched to subcellular compartments.

Rapid and robust squashed spore/colony PCR of industrially important fungi.

BACKGROUND: Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals. RESULTS: In this study we developed a rapid and robust technique called "Squash-PCR" to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains. CONCLUSION: The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.

Engineered apoptotic bodies hitchhiking across the blood-brain barrier achieved a combined photothermal-chemotherapeutic effect against glioma.

Background: Glioma as a highly lethal tumor is difficult to treat since the blood-brain barrier (BBB) restricts drug delivery into the brain. It remains a huge need for developing strategies allowing drug passage across the BBB with high efficacy. Methods: Herein, we engineered drug-loaded apoptotic bodies (Abs) loaded with doxorubicin (Dox) and indocyanine green (ICG) to cross the BBB for the treatment of glioma. The confocal laser scanning microscopy was used to characterize the structure and evaluate the hitchhiking effect of the Abs. The in vivo BBB-crossing ability and photothermal-chemotherapeutic effect of the drug-loaded Abs were investigated in mice orthotopic glioma model. Results: Engineered Abs loaded with Dox and ICG were successfully prepared. The Abs were phagocytized by macrophages, actively penetrate the BBB in vitro and in vivo utilizing the hitchhiking effect. The whole in vivo process was visualized by near-infrared fluorescence signal with a signal-to-background ratio of 7 in a mouse model of orthotopic glioma. The engineered Abs achieved a combined photothermal-chemotherapeutic effect, leading to a median survival time of 33 days in glioma-bearing mice compared to 22 days in the control group. Conclusions: This study presents engineered drug carriers with the ability to hitchhike across the BBB, providing new opportunities for the treatment of glioma.

Biomimetic Theranostic Agents with Superior NIR-II Photoacoustic and Magnetic Resonance Imaging Performance for Targeted Photothermal Therapy of Prostate Cancer.

The accurate diagnosis and treatment of prostate cancer at an early stage is crucial to reduce mortality rates. However, the limited availability of theranostic agents with active tumor-targeting abilities hinders imaging sensitivity and therapeutic efficiency. To address this challenge, we have developed biomimetic cell membrane-modified Fe2O3 nanoclusters implanted in polypyrrole (CM-LFPP), achieving photoacoustic/magnetic resonance dual-modal imaging-guided photothermal therapy of prostate cancer. The CM-LFPP exhibits strong absorption in the second near-infrared window (NIR-II, 1000-1700 nm), showing high photothermal conversion efficiency of up to 78.7% under 1064 nm laser irradiation, excellent photoacoustic imaging capabilities, and good magnetic resonance imaging ability with a T2 relaxivity of up to 48.7 s-1 mM-1. Furthermore, the lipid encapsulation and biomimetic cell membrane modification enable CM-LFPP to actively target tumors, leading to a high signal-to-background ratio of ~30.2 for NIR-II photoacoustic imaging. Moreover, the biocompatible CM-LFPP enables low-dose (0.6 W cm-2) photothermal therapy of tumors under 1064 nm laser irradiation. This technology offers a promising theranostic agent with remarkable photothermal conversion efficiency in the NIR-II window, providing highly sensitive photoacoustic/magnetic resonance imaging-guided prostate cancer therapy.

Delivery of Biomimetic Liposomes via Meningeal Lymphatic Vessels Route for Targeted Therapy of Parkinson's Disease.

Targeted therapy of Parkinson's disease is an important challenge because of the blood-brain barrier limitation. Here, we propose a natural killer cell membrane biomimetic nanocomplex (named BLIPO-CUR) delivered via the meningeal lymphatic vessel (MLV) route to further the therapeutic efficacy of Parkinson's disease. The membrane incorporation enables BLIPO-CUR to target the damaged neurons, thus improving their therapeutic efficacy through clearing reactive oxygen species, suppressing the aggregation of α-synuclein, and inhibiting the spread of excess α-synuclein species. Compared with the conventional intravenous injection, this MLV administration can enhance the delivered efficiency of curcumin into the brain by ~20 folds. The MLV route administration of BLIPO-CUR enhances the treatment efficacy of Parkinson's disease in mouse models by improving their movement disorders and reversing neuron death. Our findings highlight the great potential of MLV route administration used as targeted delivery of drugs to the brain, holding a great promise for neurodegenerative disease therapy.

A swallowable X-ray dosimeter for the real-time monitoring of radiotherapy.

Monitoring X-ray radiation in the gastrointestinal tract can enhance the precision of radiotherapy in patients with gastrointestinal cancer. Here we report the design and performance, in the gastrointestinal tract of rabbits, of a swallowable X-ray dosimeter for the simultaneous real-time monitoring of absolute absorbed radiation dose and of changes in pH and temperature. The dosimeter consists of a biocompatible optoelectronic capsule containing an optical fibre, lanthanide-doped persistent nanoscintillators, a pH-sensitive polyaniline film and a miniaturized system for the wireless readout of luminescence. The persistent luminescence of the nanoscintillators after irradiation can be used to continuously monitor pH without the need for external excitation. By using a neural-network-based regression model, we estimated the radiation dose from radioluminescence and afterglow intensity and temperature, and show that the dosimeter was approximately five times more accurate than standard methods for dose determination. Swallowable dosimeters may help to improve radiotherapy and to understand how radiotherapy affects tumour pH and temperature.

Dual-modal molecular imaging and therapeutic evaluation of coronary microvascular dysfunction using indocyanine green-doped targeted microbubbles.

Coronary microvascular dysfunction (CMD), which causes a series of cardiovascular diseases, seriously endangers human health. However, precision diagnosis of CMD is still challenging due to the lack of sensitive probes and complementary imaging technologies. Herein, we demonstrate indocyanine green-doped targeted microbubbles (named T-MBs-ICG) as dual-modal probes for highly sensitive near-infrared (NIR) fluorescence imaging and high-resolution ultrasound imaging of CMD in mouse models. In vitro results show that T-MBs-ICG can specifically target fibrin, a specific CMD biomarker, via the cysteine-arginine-glutamate-lysine-alanine (CREKA) peptide modified on the surface of microbubbles. We further employ T-MBs-ICG to achieve NIR fluorescence imaging of injured myocardial tissue in a CMD mouse model, leading to a signal-to-background ratio (SBR) of up to 50, which is 20 fold higher than that of the non-targeted group. Furthermore, ultrasound molecular imaging of T-MBs-ICG is obtained within 60 s after intravenous injection, providing molecular information on ventricular and myocardial structures and fibrin with a resolution of 1.033 mm × 0.466 mm. More importantly, we utilize comprehensive dual-modal imaging of T-MBs-ICG to evaluate the therapeutic efficacy of rosuvastatin, a cardiovascular drug for the clinical treatment of CMD. Overall, the developed T-MBs-ICG probes with good biocompatibility exhibit great potential in the clinical diagnosis of CMD.

Super-Resolution Imaging of Plant Receptor-Like Kinases Uncovers Their Colocalization and Coordination with Nanometer Resolution.

Plant cell signaling often relies on the cellular organization of receptor-like kinases (RLKs) within membrane nanodomains to enhance signaling specificity and efficiency. Thus, nanometer-scale quantitative analysis of spatial organizations of RLKs could provide new understanding of mechanisms underlying plant responses to environmental stress. Here, we used stochastic optical reconstruction fluorescence microscopy (STORM) to quantify the colocalization of the flagellin-sensitive-2 (FLS2) receptor and the nanodomain marker, remorin, within Arabidopsis thaliana root hair cells. We found that recovery of FLS2 and remorin in the plasma membrane, following ligand-induced internalization by bacterial-flagellin-peptide (flg22), reached ~85% of their original membrane density after ~90 min. The pairs colocalized at the membrane at greater frequencies, compared with simulated randomly distributed pairs, except for directly after recovery, suggesting initial uncoordinated recovery followed by remorin and FLS2 pairing in the membrane. The purinergic receptor, P2K1, colocalized with remorin at similar frequencies as FLS2, while FLS2 and P2K1 colocalization occurred at significantly lower frequencies, suggesting that these RLKs mostly occupy distinct nanodomains. The chitin elicitor receptor, CERK1, colocalized with FLS2 and remorin at much lower frequencies, suggesting little coordination between CERK1 and FLS2. These findings emphasize STORM's capacity to observe distinct nanodomains and degrees of coordination between plant cell receptors, and their respective immune pathways.

X-Ray Activatable Au/Ag Nanorods for Tumor Radioimmunotherapy Sensitization and Monitoring of the Therapeutic Response Using NIR-II Photoacoustic Imaging.

Radioimmunotherapy (RIT) is an advanced physical therapy used to kill primary cancer cells and inhibit the growth of distant metastatic cancer cells. However, challenges remain because RIT generally has low efficacy and serious side effects, and its effects are difficult to monitor in vivo. This work reports that Au/Ag nanorods (NRs) enhance the effectiveness of RIT against cancer while allowing the therapeutic response to be monitored using activatable photoacoustic (PA) imaging in the second near-infrared region (NIR-II, 1000-1700 nm). The Au/Ag NRs can be etched using high-energy X-ray to release silver ions (Ag+ ), which promotes dendritic cell (DC) maturation, enhances T-cell activation and infiltration, and effectively inhibits primary and distant metastatic tumor growth. The survival time of metastatic tumor-bearing mice treated with Au/Ag NR-enhanced RIT is 39 days compared with 23 days in the PBS control group. Furthermore, the surface plasmon absorption intensity at 1040 nm increases fourfold after Ag+ are released from the Au/Ag NRs, allowing X-ray activatable NIR-II PA imaging to monitor the RIT response with a high signal-to-background ratio of 24.4. Au/Ag NR-based RIT has minimal side effects and shows great promise for precise cancer RIT.

Low-Dose NIR-II Preclinical Bioimaging Using Liposome-Encapsulated Cyanine Dyes.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) provides a powerful tool for in vivo structural and functional imaging in deep tissue. However, the lack of biocompatible contrast agents with bright NIR-II emission has hindered its application in fundamental research and clinical trials. Herein, a liposome encapsulation strategy for generating ultrabright liposome-cyanine dyes by restricting dyes in the hydrophobic pockets of lipids and inhibiting the aggregation, as corroborated by computational modeling, is reported. Compared with free indocyanine green (ICG, an US Food and Drug Administration-approved cyanine dye), liposome-encapsulated ICG (S-Lipo-ICG) shows a 38.7-fold increase in NIR-II brightness and enables cerebrovascular imaging at only one-tenth dose over a long period (30 min). By adjusting the excitation wavelength, two liposome-encapsulated cyanine dyes (S-Lipo-ICG and S-Lipo-FD1080) enable NIR-II dual-color imaging. Moreover, small tumor nodules (2-5 mm) can be successfully distinguished and removed with S-Lipo-ICG image-guided tumor surgery in rabbit models. This liposome encapsulation maintains the metabolic pathway of ICG, promising for clinical implementation.

Albumin-Consolidated AIEgens for Boosting Glioma and Cerebrovascular NIR-II Fluorescence Imaging.

The application of an exogenous polymer matrix to construct aggregation-induced emission (AIE) nanoprobes promotes the utility of AIE luminogens (AIEgens) in diagnosing brain diseases. However, the limited fluorescence (FL) and low active-targeting abilities of AIE-based nanoprobes impede their imaging application. Here, we employed endogenous albumin as an effective matrix to encapsulate AIEgens to enhance FL quantum yield (QY) and active-targeting ability. The albumin-consolidated strategy effectively inhibited the intramolecular vibration of AIEgens and enhanced endocytosis mediated by the gp60 receptor. The QYs of three kinds of albumin-based AIE nanoprobes with FL emissions ranging from the visible (400-650 nm) to the second near-infrared (NIR-II, 1000-1700 nm) region was at least 10% higher, and the tumor-targeting efficiency was ∼25% higher, compared with those of nanoprobes constructed by the exogenous polymer. Albumin-based AIE nanoprobes have achieved active-targeting NIR-II imaging of brain tumors and cerebrovascular imaging with a high signal-to-background ratio (SBR, ∼90) and high resolution (∼70 µm) in mouse models. Therefore, the albumin-based AIE nanoprobes will enable FL imaging-guided surgery of brain tumors and cerebral ischemia, which will improve surgical efficacy to prevent recurrence and side effects.

NIR-II fluorescence visualization of ultrasound-induced blood-brain barrier opening for enhanced photothermal therapy against glioblastoma using indocyanine green microbubbles.

Focused ultrasound (FUS)-induced blood-brain barrier (BBB) opening is crucial for enhancing glioblastoma (GBM) therapies. However, an in vivo imaging approach with a high spatial-temporal resolution to monitor the BBB opening process in situ and synchronously is still lacking. Herein, we report the use of indocyanine green (ICG)-dopped microbubbles (MBs-ICG) for visualizing the FUS-induced BBB opening and enhancing the photothermal therapy (PTT) against GBM. The MBs-ICG show bright fluorescence in the second near-infrared window (NIR-II), ultrasound contrast, and ultrasound-induced size transformation properties. By virtue of complementary contrast properties, MBs-ICG can be successfully applied for cerebral vascular imaging with NIR-II fluorescence resolution of ∼168.9 µm and ultrasound penetration depth of ∼7 mm. We further demonstrate that MBs-ICG can be combined with FUS for in situ and synchronous visualization of the BBB opening with a NIR-II fluorescence signal-to-background ratio of 6.2 ± 1.2. Finally, our data show that the MBs-ICG transform into lipid-ICG nanoparticles under FUS irradiation, which then rapidly penetrate the tumor tissues within 10 min and enhance PTT in orthotopic GBM-bearing mice. The multifunctional MBs-ICG approach provides a novel paradigm for monitoring BBB opening and enhancing GBM therapy.

Near Infrared Emitting Semiconductor Polymer Dots for Bioimaging and Sensing.

Semiconducting polymer dots (Pdots) are rapidly becoming one of the most studied nanoparticles in fluorescence bioimaging and sensing. Their small size, high brightness, and resistance to photobleaching make them one of the most attractive fluorophores for fluorescence imaging and sensing applications. This paper highlights our recent advances in fluorescence bioimaging and sensing with nanoscale luminescent Pdots, specifically the use of organic dyes as dopant molecules to modify the optical properties of Pdots to enable deep red and near infrared fluorescence bioimaging applications and to impart sensitivity of dye doped Pdots towards selected analytes. Building on our earlier work, we report the formation of secondary antibody-conjugated Pdots and provide Cryo-TEM evidence for their formation. We demonstrate the selective targeting of the antibody-conjugated Pdots to FLAG-tagged FLS2 membrane receptors in genetically engineered plant leaf cells. We also report the formation of a new class of luminescent Pdots with emission wavelengths of around 1000 nm. Finally, we demonstrate the formation and utility of oxygen sensing Pdots in aqueous media.

Miniature NIR-II Nanoprobes for Active-Targeted Phototheranostics of Brain Tumors.

Nanoprobes (NPs) in the second near-infrared biowindow (NIR-II, 1000-1700 nm) are developed and widely used in cancer phototheranostics. However, most NIR-II NPs exhibit low phototheranostic efficiency due to their tedious synthetic routes, large particle sizes (>20 nm), and lack of active targeting properties. Here, miniature NIR-II NPs, named HSA-ICG-iRGD, for active-targeted NIR-II phototheranostics of brain tumors are reported. The HSA-ICG-iRGD probes are designed based on hydrophobic interactions as well as hydrogen bonds between albumin and indocyanine green derivatives (ICG-iRGD) via molecular docking. The as-prepared NPs have a compact size of 10 nm and show tumor-targeting ability by specifically binding to αv ß3 integrin receptors which are highly expressed on the surface of brain tumor cells via iRGD peptides. The HSA-ICG-iRGD NPs are then applied to perform active-targeted NIR-II fluorescence imaging, resulting in a signal-to-background ratio of 6.85 in orthotopic glioma mouse models. Under the selected laser irradiation of 808 nm, the photothermal effect of HSA-ICG-iRGD extends the survival of the tumor-bearing mice to 55 days, significantly longer than that of the control group (30 days). These results highlight the potential of miniature NPs for active-targeted NIR-II fluorescence imaging and phototherapy of brain tumors.

Gold Nanoclusters-Based NIR-II Photosensitizers with Catalase-like Activity for Boosted Photodynamic Therapy.

Photodynamic therapy (PDT) under fluorescence imaging as a selective and non-invasive treatment approach has been widely applied for the therapy of cancer and bacterial infections. However, its treatment efficiency is hampered by high background fluorescence in the first near-infrared window (NIR-I, 700-900 nm) and oxygen-dependent photosensitizing activity of traditional photosensitizers. In this work, we employ gold nanoclusters (BSA@Au) with the second near-infrared (NIR-II, 1000-1700 nm) fluorescence and catalase-like activity as alternative photosensitizers to realize highly efficient PDT. The bright NIR-II fluorescence of BSA@Au enables the visualization of PDT for tumor with a high signal-to-background ratio (SBR = 7.3) in 4T1 tumor-bearing mouse models. Furthermore, the catalase-like activity of BSA@Au endows its oxygen self-supplied capability, contributing to a five-fold increase in the survival period of tumor-bearing mice receiving boosted PDT treatment compared to that of the control group. Moreover, we further demonstrate that BSA@Au-based PDT strategy can be applied to treat bacterial infections. Our studies show the great potential of NIR-II BSA@Au as a novel photosensitizer for boosted PDT against cancer and bacterial infections.

Hydroporphyrin-Doped Near-Infrared-Emitting Polymer Dots for Cellular Fluorescence Imaging.

Near-infrared (NIR) fluorescent semiconductor polymer dots (Pdots) have shown great potential for fluorescence imaging due to their exceptional chemical and photophysical properties. This paper describes the synthesis of NIR-emitting Pdots with great control and tunability of emission peak wavelength. The Pdots were prepared by doping poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1',3)-thiadiazole)] (PFBT), a semiconducting polymer commonly used as a host polymer in luminescent Pdots, with a series of chlorins and bacteriochlorins with varying functional groups. Chlorins and bacteriochlorins are ideal dopants due to their high hydrophobicity, which precludes their use as molecular probes in aqueous biological media but on the other hand prevents their leakage when doped into Pdots. Additionally, chlorins and bacteriochlorins have narrow deep red to NIR-emission bands and the wide array of synthetic modifications available for modifying their molecular structure enables tuning their emission predictably and systematically. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements show the chlorin- and bacteriochlorin-doped Pdots to be nearly spherical with an average diameter of 46 ± 12 nm. Efficient energy transfer between PFBT and the doped chlorins or bacteriochlorins decreases the PFBT donor emission to near baseline level and increases the emission of the doped dyes that serve as acceptors. The chlorin- and bacteriochlorin-doped Pdots show narrow emission bands ranging from 640 to 820 nm depending on the doped dye. The paper demonstrates the utility of the systematic chlorin and bacteriochlorin synthesis approach by preparing Pdots of varying emission peak wavelength, utilizing them to visualize multiple targets using wide-field fluorescence microscopy, binding them to secondary antibodies, and determining the binding of secondary antibody-conjugated Pdots to primary antibody-labeled receptors in plant cells. Additionally, the chlorin- and bacteriochlorin-doped Pdots show a blinking behavior that could enable their use in super-resolution imaging methods like STORM.

Endoscopic Ultrasound Localization Microscopy for the Evaluation of the Microvasculature of Gastrointestinal Tract Tumors in Rabbits.

OBJECTIVE: The morphological and hemodynamic characterization of the microvascular network around the gastrointestinal (GI) tract can be of significant clinical value for the early diagnosis and treatment of GI tract cancer. Ultrasound localization microscopy (ULM) imaging has been demonstrated to be capable of resolving the microvascular network. However, the endoscopic application of ULM imaging techniques is still unknown. In this study, an endoscopic ultrasound localization microscopy (e-ULM) imaging technique was developed to evaluate the changes of microvasculature during GI tract tumor growth. METHODS: A customized circular array transducer (center frequency: 6.8 MHz) and the coherent diverging wave compounding method were used to generate B-mode images. Spatiotemporal singular value decomposition processing was used to eliminate the background signals before signal localizations. The centroids of spatially isolated signals were localized and summed to generate the final super-resolution image. RESULTS: The final microvasculature map of a rabbit GI tract tumor reveals that e-ULM can be used to surpass the diffraction limit in traditional endoscopic ultrasound (EUS) imaging. Furthermore, it is observed that data from different stages of tumor growth exhibit significant differences in microvascular pattern and density. CONCLUSION: Our study demonstrated the implementation and application of an in vivo e-ULM imaging technique for the evaluation of the microvasculature of GI tumors. SIGNIFICANCE: The efficient e-ULM imaging technique shows potential for use in the detection of GI tract tumor microcirculation changes and subsequent diagnosis of GI tract cancer.