Cell Imaging Research Articles

Imaging of tumor-associated macrophage dynamics during immunotherapy using a CD163-specific nanobody-based immunotracer

Immunotherapies have emerged as an effective treatment option for immune-related diseases, such as cancer and inflammatory diseases. However, variations in patient responsiveness limit the broad applicability and success of these immunotherapies. Noninvasive whole-body imaging of the immune status of individual patients during immunotherapy could enable the prediction and monitoring of the patient's response, resulting in more personalized treatments. In this study, we developed a nanobody-based immunotracer targeting CD163, a receptor specifically expressed on macrophages. This anti-CD163 immunotracer bound to human and mouse CD163 with high affinity and specificity without competing for ligand binding. Furthermore, the tracer showed no unwanted immune cell activation and was nonimmunogenic. Upon radiolabeling of the anti-CD163 immunotracer, specific imaging of CD163+ macrophages using micro-single-photon emission computerized tomography/computed tomography or micro-positron emission tomography/CT was performed. The anti-CD163 immunotracer was able to stratify immunotherapy responders from nonresponders (NR) by visualizing differences in the intratumoral CD163+ TAM distribution in Lewis lung carcinoma-ovalbumin tumor-bearing mice receiving an anti-programmed cell death protein-1 (PD-1)/CSF1R combination treatment. Immunotherapy-responding mice showed a more homogeneous distribution of the PET signal in the middle of the tumor, while CD163+ TAMs were located at the tumor periphery in NR. As such, visualization of CD163+ TAM distribution in the tumor microenvironment could allow a prediction or follow-up of therapy response. Altogether, this study describes an immunotracer, specific for CD163+ macrophages, that allows same-day imaging and follow-up of these immune cells in the tumor microenvironment, providing a good basis for the prediction and follow-up of immunotherapy responses in cancer patients.

PFKFB3-dependent redox homeostasis and DNA repair support cell survival under EGFR-TKIs in non-small cell lung carcinoma

BackgroundThe efficacy of tyrosine kinase inhibitors (TKIs) targeting the EGFR is limited due to the persistence of drug-tolerant cell populations, leading to therapy resistance. Non-genetic mechanisms, such as metabolic rewiring, play a significant role in driving lung cancer cells into the drug-tolerant state, allowing them to persist under continuous drug treatment.MethodsOur study employed a comprehensive approach to examine the impact of the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) on the adaptivity of lung cancer cells to EGFR TKI therapies. We conducted metabolomics to trace glucose rerouting in response to PFKFB3 inhibition during TKI treatment. Live cell imaging and DCFDA oxidation were used to quantify levels of oxidation stress. Immunocytochemistry and Neutral Comet assay were employed to evaluate DNA integrity in response to therapy-driven oxidative stress.ResultsOur metabolic profiling revealed that PFKFB3 inhibition significantly alters the metabolic profile of TKI-treated cells. It limited glucose utilization in the polyol pathway, glycolysis, and TCA cycle, leading to a depletion of ATP levels. Furthermore, pharmacological inhibition of PFKFB3 overcome TKI-driven redox capacity by diminishing the expression of glutathione peroxidase 4 (GPX4), thereby exacerbating oxidative stress. Our study also unveiled a novel role of PFKFB3 in DNA oxidation and damage by controlling the expression of DNA-glycosylases involved in base excision repair. Consequently, PFKFB3 inhibition improved the cytotoxicity of EGFR-TKIs by facilitating ROS-dependent cell death.ConclusionsOur results suggest that PFKFB3 inhibition reduces glucose utilization and DNA damage repair, limiting the adaptivity of the cells to therapy-driven oxidative stress and DNA integrity insults. Inhibiting PFKFB3 can be an effective strategy to eradicate cancer cells surviving under EGFR TKI therapy before they enter the drug-resistant state. These findings may have potential implications in the development of new therapies for drug-resistant cancer treatment.

Insights into Cancer Cell Imaging Probes Based on Chalcone Scaffolds: Theoretical and Experimental Perspectives.

The research article details the synthesis of chalcone-chromone-based scaffolds via multicomponent reactions. These compounds were characterized using conventional spectroscopic methods, including NMR (1H and 13C), FT-IR, and HR-MS. Among the synthesized scaffolds, AZBNPy stood out, exhibiting exceptional DNA and protein targeting capabilities with superior binding parameters. Molecular docking studies indicated that AZBNPy has potential as a potent anticancer agent and a probe for cancer cell imaging. The findings showed that AZBNPy effectively inhibited cell proliferation and induced cell death by targeting HER-2, PARP-2, and HFR in MCF-7 cells. Additionally, in vitro fluorescence imaging studies confirmed AZBNPy's specificity for cancer cell receptors, displaying strong fluorescence in human breast cancer tissues. The clinical application of AZBNPy as an optical imaging agent holds significant promise in aiding surgeons with the precise identification and removal of cancerous tissues, potentially improving patient outcomes and survival rates.

Identifying the composition of large vesicles in the cytoplasm of oocytes.

Context Oocyte vesicles, or vacuoles, have been described using transmission electron microscopy in most species. In sheep and cow oocytes, vesicles constitute up to 30% of the cytoplasm, their volume decreases during maturation and is lower in poorer quality oocytes, suggesting they are important for oocyte competence. However, the composition and function of these organelles is unknown. Aim This study aimed to ascertain the content of oocyte vesicles and examine the effect of different fixation methods on the size and preservation of these organelles. Methods Sheep oocytes were centrifuged to segregate organelles then stained with organelle-specific fluorescent dyes (Nile Red, LysoTracker, Fluo-4-AM and TMRM) and imaged by live cell confocal microscopy. The oocytes were fixed with either glutaraldehyde or paraformaldehyde and prepared for electron microscopy to confirm the distribution of organelles and compare ultrastructure and organelle size. Key results Nile Red staining has identified that vesicles contain lipid that is different to that in the osmium-stained lipid droplets observed by electron microscopy. Lipid droplets and vesicles were significantly smaller when prepared for electron microscopy compared to live cell imaging. Organelles were less likely to be fully segregated following centrifugation in oocytes prior to maturation (20%) compared to oocytes after maturation (77%; P Conclusions Oocyte vesicles are lipid storing organelles that may be important for oocyte quality. Implications This study highlights the importance of lipid for oocyte quality and the need for further research to identify the optimal fatty acid content for in vitro maturation media and oocyte competence.

Live Cell Imaging of Human Liver Fibrosis using Hepatic Micro-Organoids.

Due to the limitations of available in vitro systems and animal models, we lack a detailed understanding of the pathogenetic mechanisms and have minimal treatment options for liver fibrosis. Therefore, we engineered a live cell imaging system that assesses fibrosis in a human multi-lineage hepatic organoid in a microwell (i.e., microHOs). Transcriptomic analysis revealed that TGFβ1 converted mesenchymal cells in microHOs into myofibroblast-like cells resembling those in fibrotic human liver tissue. When pro-fibrotic intracellular signaling pathways were examined, the anti-fibrotic effect of receptor-specific tyrosine kinase inhibitors was limited to the fibrosis induced by the corresponding growth factor, which indicates their anti-fibrotic efficacy would be limited to fibrotic diseases solely mediated by that growth factor. Based upon transcriptomic and transcription factor activation analyses in microHOs, GSK3β and p38 MAPK inhibitors were identified as potential new broad-spectrum therapies for liver fibrosis. Other new therapies could subsequently be identified using the microHO system.

Preclinical and Clinical-Scale Magnetic Particle Imaging of Natural Killer Cells: in vitro and ex vivo Demonstration of Cellular Sensitivity, Resolution, and Quantification.

Clinical adoption of NK cell immunotherapy is underway for medulloblastoma and osteosarcoma, however there is currently little feedback on cell fate after administration. We propose magnetic particle imaging (MPI) may have applications for the quantitative detection of NK cells. Human-derived NK-92 cells were labeled by co-incubation with iron oxide nanoparticles (VivoTrax™) for 24h then excess nanoparticles were washed with centrifugation. Cytolytic activity of labeled versus unlabeled NK-92 cells was assessed after 4h of co-incubation with medulloblastoma cells (DAOY) or osteosarcoma cells (LM7 or OS17). Labeled NK-92 cells at two different doses (0.5 or 1 × 106) were administered to excised mouse brains (cerebellum), fibulas, and lungs then imaged by 3D preclinical MPI (MOMENTUM™) for detection relative to fiducial markers. NK-92 cells were also imaged by clinical-scale MPI under development at Magnetic Insight Inc. NK-92 cells were labeled with an average of 3.17 pg Fe/cell with no measurable effects on cell viability or cytolytic activity against 3 tumor cell lines. MPI signal was directly quantitative with the number of labeled NK-92 cells, with preclinical limit of detection of 3.1 × 104 cells on MOMENTUM imager. Labeled NK-92 cells could be accurately localized in mouse brains, fibulas, and lungs within < 1mm of stereotactic injection coordinates with preclinical scanner. Feasibility for detection on a clinical-scale MPI scanner was demonstrated using 4 × 107 labeled NK-92 cells, which is in the range of NK cell doses administered in our previous clinical trial. MPI can provide sensitive, quantitative, and accurate spatial information on NK cells soon after delivery, showing initial promise to address a significant unmet clinical need to track NK cell fate in patients.

MRNA-encoded fluorescent proteins enable superior organelle labeling for live cell imaging.

Live cell labeling of various organelles is of great demand in the research field of cell biology. However, current approaches often lack an optimal balance between efficiency and versatility. We took advantage of chemical modified mRNA to express various organelle located fluorescent proteins. This approach facilitated highly efficient multi-organelle labeling across diverse cell types, both invitro and invivo. The application of mRNA-based organelle markers offers faster and more efficient transfection and expression compared to plasmids. When expressing fluorescent proteins in neuronal cells, it is faster and safer compared to viral vectors. Notably, this approach excels in rapidly labeling organelles, making it highly effective for dynamic live cell imaging applications. By leveraging this tool, we unveiled the unique behaviors of mitochondria and the endoplasmic reticulum during spike protein-mediated cell fusion, a critical event in SARS-CoV-2 viral entry. Thus, our results demonstrate the potency of mRNA-encoded fluorescent proteins as powerful tools for advancing biological research.

DRIM modulates Src activation and regulates angiogenic functions in vascular endothelial cells.

Downregulated in Metastasis Protein (DRIM) was discovered in malignant epithelial cells and was thought to be mainly a nucleus protein affecting cancer cells. Recent single-cell sequencing analysis suggests that DRIM is abundantly expressed in vascular endothelial cells. There has been no knowledge of the role of DRIM in the endothelium. In the present study, using protein fraction method and cell imaging, we identified that the DRIM protein was abundantly present in both nucleus and the cytoskeletal fractions of human vascular endothelial cells. Knockdown of DRIM in the endothelial cells significantly affected growth, migration, and angiogenic tubule formation. Proteomics analyses revealed that Src was an important direct target protein of DRIM, a finding further confirmed by protein interaction assay. Silencing DRIM activated the tyrosine 419 site phosphorylation of Src kinase in endothelial cells, thereby affecting the downstream proteins of Src including p-FAK and p-STAT3, and exerting biological effects. To conclude, our results provide evidence of DRIM being a nuclear and cytoskeletal-associated protein, having a novel key role of the protein in vascular endothelial cells.

Red-light Emitting Orthogonally Trireactive Gold Nanoclusters for the Synthesis of Multifunctionalized Nanomaterials.

For the development of highly multifunctionalized nanomaterials, the introduction of functional molecules on gold nanoclusters containing thiols preinstalled with connecting groupsconstitutes a promising approach. However, the uniform introduction of multiple connecting groups while avoiding side reactions is a challenging task. Herein, the synthesis of gold nanoclusters (ca. 1nm) coated with thiol peptides bearing azido, amino, and aminooxy groups is reported. These nanoclusters emit red-light before and after the functionalization, enabling application to cell imaging. A detailed structure analysis using transmission electron microscope and X-ray absorption spectroscopy reveals the formation of Aun(SR)m nanoclusters as promising motifs for red-light emission. The sequential modification of the trireactive nanoclusters with RGD (Arg-Gly-Asp) peptides, metal chelators, and anticancer drugs via [3+2] cycloaddition, oximation, and amidation reactions at each functional group furnish red-light-emissive nanomaterials exhibiting remarkable toxicity against A549 human lung cancer cells. Integration of the multiligation chemistry and gold nanocluster engineering pave the way toward the development of advanced multifunctional nanomaterials for biological applications.

Cutting Edge Microscopic Intraoperative Tissue Assessment for Guidance in Oncologic Surgery: A Systematic Review of the Role of Optical Coherence Tomography.

There is a demand for intraoperative diagnostic support and image guidance in oncological surgery. Novel techniques can provide images similar to histopathological slides within a few minutes. Optical coherence tomography (OCT) and full-field OCT (FF-OCT) provide images with resolution greater than a hundred micrometers without the need for exogenous contrast agents or specimen staining. The aim of this systematic review was to assess the current clinical applications of OCT and dynamic cell imaging (DCI) in oncologic surgery, examining the translation challenges and proposing perspectives for improving future clinical applications. The study adhered to the PRISMA guidelines. PubMed, Google Scholar, and ClinicalTrials.gov were searched up to July 2024. Manuscripts reporting data on OCT and (D)-FF-OCT application in oncologic surgery were included in the qualitative analysis. Thirty-one studies met the inclusion criteria. Most were from the fields of dermatologic (25.8%) and breast cancer (29%) surgery, followed by prostate and bladder (9.6%), ovarian (9.6%), head and neck (6.4%), gastrointestinal (6.4%), hepato-biliary (3.2%), and general surgery (9.6%). The majority of articles focused on FF-OCT and DCI (80.6%). Compared with the gold standard of final pathology, the OCT sensitivity ranged between 66.7 and 94%, the specificity between 64 and 100%, and the accuracy between 73 and 96%. The medical use of OCT has expanded from ophtalmology to other fields including gastroenterology and oncology and, with techniques such as FF-OCT and DCI, can enable rapid intraoperative diagnosis beyond classic histopathology.

ChromTR: chromosome detection in raw metaphase cell images via deformable transformers.

Chromosome karyotyping is a critical way to diagnose various hematological malignancies and genetic diseases, of which chromosome detection in raw metaphase cell images is the most critical and challenging step. In this work, focusing on the joint optimization of chromosome localization and classification, we propose ChromTR to accurately detect and classify 24 classes of chromosomes in raw metaphase cell images. ChromTR incorporates semantic feature learning and class distribution learning into a unified DETR-based detection framework. Specifically, we first propose a Semantic Feature Learning Network (SFLN) for semantic feature extraction and chromosome foreground region segmentation with object-wise supervision. Next, we construct a Semantic-Aware Transformer (SAT) with two parallel encoders and a Semantic-Aware decoder to integrate global visual and semantic features. To provide a prediction with a precise chromosome number and category distribution, a Category Distribution Reasoning Module (CDRM) is built for foreground-background objects and chromosome class distribution reasoning. We evaluate ChromTR on 1404 newly collected R-band metaphase images and the public G-band dataset AutoKary2022. Our proposed ChromTR outperforms all previous chromosome detection methods with an average precision of 92.56% in R-band chromosome detection, surpassing the baseline method by 3.02%. In a clinical test, ChromTR is also confident in tackling normal and numerically abnormal karyotypes. When extended to the chromosome enumeration task, ChromTR also demonstrates state-of-the-art performances on R-band and G-band two metaphase image datasets. Given these superior performances to other methods, our proposed method has been applied to assist clinical karyotype diagnosis.

Dynamic cell imaging by iterative phase retrieval microscopy

Dynamic cell imaging by iterative phase retrieval microscopy

In vivo scanning laser fundus and high-resolution OCT imaging of retinal ganglion cell injury in a non-human primate model with an activatable fluorescent-labeled TAT peptide probe.

The optical imaging agent TcapQ488 has enabled imaging of retinal ganglion cell (RGC) injury in vivo in rodents and has potential as an effective diagnostic probe for early detection and intervention monitoring in glaucoma patients. In the present study, we investigated TcapQ488 in non-human primates (NHPs) to identify labeling efficacy and early signals of injured RGC, to determine species-dependent changes in RGC probe uptake and clearance, and to determine dose-limiting toxicities. Doses of 3, 6, and 12 nmol of TcapQ488 were delivered intravitreally to normal healthy NHP eyes and eyes that had undergone hemiretinal endodiathermy axotomy (HEA) in the inferior retina. Post-injection fundus fluorescence imaging using a Spectralis imaging platform (Heidelberg Engineering) documented TcapQ488 activation in RGC cell bodies. Optical coherence tomography (OCT), slit-lamp examinations, intraocular pressure measurements, and visual electrophysiology testing were performed to monitor probe tolerability. For comparison, a negative control, non-cleavable, non-quenched probe (dTcap488, 6 nmol), was delivered intravitreally to a normal healthy eye. In normal healthy eyes, intravitreal injection of 3 nmol of TcapQ488 was well-tolerated, while 12 nmol of TcapQ488 to the healthy eye caused extensive probe activation in the ganglion cell layer (GCL) and eventual retinal nerve fiber layer thinning. In HEA eyes, the HEA procedure followed by intravitreal TcapQ488 (3 nmol) injection resulted in probe activation within cell bodies in the GCL, confined to the HEA-treated inferior retina, indicating cell injury and slow axonal transport in the GCL. However, in contrast to rodents, a vitreal haze that lasted 2-12 weeks obscured rapid high-resolution imaging of the fundus. By contrast, intravitreal TcapQ488 injection prior to the HEA procedure led to minimal probe labeling in the GCL. The results of the dTcap488 control experiments indicated that fast axonal transport carried the probe out of the retina after cell body uptake. No evidence of pan-retinal toxicity or loss of retino-cortical function was detected in any of the three NHPs tested. Overall, these data provide evidence of TcapQ488 activation, without toxicity, in NHP HEA eyes that had been intravitreally injected with 3 nmol of the probe. Compared to rodents, unexpectedly rapid axonal transport in the NHPs reduced the capacity to visualize RGC cell bodies and axons through the backdrop of an intravitreal haze. Nonetheless, although intravitreal clearance rates did not scale to NHPs, HEA-induced reductions in axonal transport enhanced probe visualization in the cell body.

A conserved bacterial genetic basis for commensal-host specificity.

Animals selectively acquire specific symbiotic gut bacteria from their environments that aid host fitness. To colonize, a symbiont must locate its niche and sustain growth within the gut. Adhesins are bacterial cell surface proteins that facilitate attachment to host tissues and are often virulence factors for opportunistic pathogens. However, the attachments are often transient and nonspecific, and additional mechanisms are required to sustain infection. In this work, we use live imaging of individual symbiotic bacterial cells colonizing the gut of living Drosophila melanogaster to show that Lactiplantibacillus plantarum specifically recognizes the fruit fly foregut as a distinct physical niche. L. plantarum establishes stably within its niche through host-specific adhesins encoded by genes carried on a colonization island. The adhesin binding domains are conserved throughout the Lactobacillales, and the island also encodes a secretion system widely conserved among commensal and pathogenic bacteria.

Chimeric antigen receptor macrophages targeting c-MET(CAR-M-c-MET) inhibit pancreatic cancer progression and improve cytotoxic chemotherapeutic efficacy

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors. Macrophages are abundant in the tumor microenvironment, making them an attractive target for therapeutic intervention. While current immunotherapies, including immune checkpoint inhibition (ICI) and chimeric antigen receptor T (CAR-T) cells, have shown limited efficacy in pancreatic cancer, a novel approach involving chimeric antigen receptor macrophages (CAR-M) has, although promising, not been explored in pancreatic cancer. In this study, we first investigated the role of CAR-M cells targeting c-MET in pancreatic cancer.MethodsThe effectiveness and rationality of c-MET as a target for CAR-M in pancreatic cancer were validated through bioinformatic analyses and immunohistochemical staining of samples from pancreatic cancer patients. We utilized flow cytometry and bioluminescence detection methods to demonstrate the specific binding and phagocytic killing effect of CAR-M on pancreatic cancer cells. Additionally, we observed the process of CAR-M engulfing pancreatic cancer cells using confocal microscopy and a long-term fluorescence live cell imaging system. In an in situ tumor model transplanted into NOD/SCID mice, we administered intraperitoneal injections of CAR-M to confirm its inhibitory function on pancreatic cancer. Furthermore, we validated these findings in human monocyte-derived macrophages (hMDM).ResultsBioinformatics and tumor tissue microarray analyses revealed significantly higher expression levels of c-MET in tumor tissues, compared to the paired peritumoral tissues, and higher c-MET expression correlated with worse patient survival. CAR-M cells were engineered using human monocytic THP-1 cell line and hMDM targeting c-MET (CAR-M-c-MET). The CAR-M-c-MET cells demonstrated highly specific binding to pancreatic cancer cells and exhibited more phagocytosis and killing abilities than the pro-inflammatory polarized control macrophages. In addition, CAR-M-c-MET cells synergized with various cytotoxic chemotherapeutic drugs. In a NOD/SCID murine model, intraperitoneally injected CAR-M-c-MET cells rapidly migrated to tumor tissue and substantially inhibited tumor growth, which did not lead to obvious side effects. Cytokine arrays and mRNA sequencing showed that CAR-M-c-MET produced higher levels of immune activators than control macrophages.ConclusionsThis study provides compelling evidence for the safety and efficacy of CAR-M therapy in treating pancreatic cancer. The results demonstrate that CAR-M-c-MET significantly suppresses pancreatic cancer progression and enhances the effectiveness of cytotoxic chemotherapy. Remarkably, no discernible side effects occur. Further clinical trials are warranted in human pancreatic cancer patients.

Curcumin-Derived (3-Aminopropyl)trimethoxysilane-Functionalized Carbon Quantum Dots: A Fluorometric and Colorimetric Nanoprobe for Picric Acid Detection, Antioxidant Activity, and Liposome Encapsulation.

Creating an analytical probe to track extremely mutagenic picric acid (PA) is essential for human health and the environment. Here, we developed a straightforward and quick fluorescence analytical method utilizing 3-aminopropyltrimethoxysilane (3-APTMS)-functionalized curcumin carbon quantum dots (CQDs) for the fast and selective detection of PA. Solvothermal carbonization and functionalization of curcumin with 3-APTMS were used to create multifunctional CQDs, which were then characterized using UV-vis spectroscopy, Fourier transform infrared (FTIR), X-ray diffraction (XRD), ζ-potential, transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). Our CQDs, as synthesized with an average diameter of 3.4 nm, exhibited excitation-dependent emission behavior, demonstrating 63.85% yield, 1.59% quantum yield, and fluorescence lifetime decay broader than a single exponential. The addition of picric acid significantly reduced the fluorescence (FL) emission intensity of CQDs and caused a noticeable color shift in visible as well as UV light. Throughout the 0.1-2.5 μM range, the calibration curve of the suggested assay demonstrated favorable linearity between quenched FL emission intensity and PA concentration, with the lowest detection limit of 88.96 nM. The CQD shows antioxidant activity at low concentrations (<0.07 mg/mL), measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (colorimetry and electrochemically). Further, we encapsulated our CQDs in the liposome to make it biocompatible for cell imaging for future study. The results indicate the efficacy of CQDs as a nanoprobe for the selective detection of PA, retaining a few of the primary properties of natural curcumin-like antioxidant activity while having significantly higher bioavailability and water solubility; they can be used as a modifier in semiconductors for photocatalytic application and can also be a promising fluorescence probe in cell imaging.

Photocurrent Polarity-Switchable Imaging of Single Living Cells by Light-Addressable Electrochemical Sensor.

The light-addressable electrochemical sensor (LAES) is a powerful tool for single-cell imaging due to its label-free and probe-free advantages. In this work, we report a photocurrent polarity-switchable LAES using a single-phase photoelectrode of a BiFeO3 thin film for living cell imaging. The proposed BiFeO3 could show both p- and n-type photocurrent behavior by simply altering the external bias voltage. LAES imaging of the same individual MCF-7 cells was performed in anodic and cathodic modes. Decreases in both photocurrents were observed due to the hindering effect of the adherent cells on local photoinduced Faraday currents. Furthermore, the dynamic photocurrent changes on cells after trypsin treatment were imaged and studied at anode and cathode polarities. Both polarities showed an increase in local photocurrents on cells as the cell-substrate junction weakened, and this change displayed heterogeneous characteristics. This is the first time that LAES cell imaging was achieved in a p-type mode. Meanwhile, our photocurrent polarity-switchable imaging approach overcomes the limitations of conventional photoelectrodes, which have been confined to single-polarity operation. We believe this work has the potential to significantly broaden the application scope of LAES in single-cell visualization and analysis, offering great insights into cellular behavior and function.

Tunable Nano-Supramolecules Based on Cucurbiturils for Near-Infrared Phosphorescence Imaging.

Nano-supramolecules based on artificial macrocycles can not only regulate assembly morphology but also boost phosphorescence resonance energy transfer (PRET). Herein, a water-soluble phosphorescence supramolecule was constructed from the hyaluronic acid-modified bromophenylpyridinium (HAPY), cucurbit[n]uril (CB[n], n = 7/8), and energy acceptor phenyl-bridged phenothiazine derivatives, displaying efficient PRET and achieving near-infrared (NIR) phosphorescence by macrocyclic CB[n] and the assembly confinements. As compared with weak phosphorescent nanofibers of HAPY/CB[7], the spherical nanoparticles of HAPY/CB[8] not only gave strong green phosphorescence with extended lifetime to 1.27 ms but also could act as the energy donor and confine cationic phenothiazine in the secondary assemblies, leading to highly efficient PRET efficiency (87.27%) from the phosphors to triplet acceptors, realizing phosphorescence emission at 750 nm and an ultralarge Stokes shift of 440 nm. Ultimately, the nanoassembly achieved by the multiscale confinements boosting PRET was successfully applied in targeted cancer cell imaging, providing new insight for fabricating NIR phosphorescence materials.

Design and synthesis of piperine-based photoaffinity probes for revealing potential targets of piperine in neurological disease.

Piperine (PIP) has attracted extensive attention due to its diverse biological activities. In this study, we developed two photoaffinity probes PIP-1 and PIP-2, which are biologically safe and retain PIP's bioactivity, to investigate its protein targets in vivo. Using in situ labeling and cell imaging, we were able to effectively detect and visualize the drug targets of PIP with our probes. Additionally, a series of protein targets of PIP were fished using PIP-2 through proteome profiling, with further validation suggesting that TGFβ1 might be a potential target involved in PIP's effects on neurological diseases. These findings demonstrate that PIP-2 is a valuable tool for identifying the targets of PIP.

Low-Intensity Pulsed Ultrasound Dynamically Modulates the Migration of BV2 Microglia

ObjectiveLow-intensity pulsed ultrasound (LIPUS) is a promising modality for neuromodulation. Microglia are the resident immune cells in the brain and their mobility is critical for maintaining brain homeostasis and alleviating neuroimmune pathologies. However, it is unclear whether and how LIPUS modulates microglial migration in physiological conditions. MethodsHere we examined the in vitro effects of LIPUS on the mobility of BV2 microglia by live cell imaging. Single-cell tracing of BV2 microglia migration was analyzed using ImageJ and Chemotaxis and Migration Tool software. Pharmacological manipulation was performed to determine the key molecular players involved in regulating ultrasound-dependent microglia migration. ResultsWe found that the distance of microglial migration was enhanced by LIPUS with increasing acoustic pressure. Removing the extracellular Ca2+ influx or depletion of intracellular Ca2+ stores suppressed ultrasound-enhanced BV2 migration. Furthermore, we found that blocking the reorganization of actin, or suppressing purinergic signaling by application of apyrase or hemi-channel inhibitors, both diminished ultrasound-induced BV2 migration. LIPUS stimulation also enhanced microglial migration in a lipopolysaccharide (LPS)-induced inflammatory environment. ConclusionLIPUS promoted microglia migration in both physiological and inflammatory environments. Calcium, cytoskeleton, and purinergic signaling were involved in regulating ultrasound-dependent microglial mobility. Our study reveals the biomechanical impact of ultrasound on microglial migration and highlights the potential of using ultrasound-based tools for modulation of microglial function.