We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,...We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,a conventional two-photon microscope was equipped with a 3D-printed holders,which stabilize the tissue surface within the focal plane of immersion objectives.Further computational image stabilization and noise reduction were applied,followed by superresolution radial fluctuations(SRRF)analysis,doubling image resolution,and enhancing signal-to-noise ratios for in vivo subcellular process investigation.Stabilization of<1μm was obtained by suction,and<25 nm were achieved by subsequent algorithmic image stabilization.A Mito-Dendra2 mouse model,expressing green fluorescent protein(GFP)in mitochondria,demonstrated the potential of long-term intravital subcellular imaging.In vivo mitochondrial fission and fusion,mitochondrial status migration,and the effects of alcohol consumption(modeled as an alcoholic liver disease)and berberine treatment on hepatocyte mitochondrial dynamics are directly observed intravitally.Suction-based stabilization in two-photon intravital imaging,coupled with computational super-resolution holds promise for advancing in vivo subcellular imaging studies.展开更多
AIM： To investigate microvascular injury quantitatively in the small bowel with respect to cardiopulmonary bypass （CPB） and related mechanisms. METHODS： In 10 male SD rats, normothermic CPB was established and con...AIM： To investigate microvascular injury quantitatively in the small bowel with respect to cardiopulmonary bypass （CPB） and related mechanisms. METHODS： In 10 male SD rats, normothermic CPB was established and continued with a flow rate of 100-150 mL/kg per minute for 60 min, while another 10 sham-operated animals served as controls. An approximate 10-cm loop of the terminal ileum was exteriorized for observation by means of intravital fluorescence microscopy. The small bowel microcirculatory network including arterioles, capillaries, and collecting venules was observed prior to CPB, CPB 30 min, CPB 60 min, post-CPB 60 min and post-CPB 120 rain. The intestinal capillary perfusion, microvascular permeability and leukocyte adherence were also measured. RESULTS： The systemic hemodynamics remained stable throughout the experiment in both groups. In CPB animals, significant arteriolar vasoconstriction, blood velocity reduction and functional capillary density diminution were found. As concomitances, exaggerated albumin extravasation and increased leukocyte accumulation were also noted. These changes were more pronounced and there were no signs of restitution at the end of the observation period. CONCLUSION： CPB induces significant microcirculatory injury of the small bowel in rats. The major underlying mechanisms are blood flow redistribution and generalized inflammatory response associated with CPB.展开更多
CD8＋ T cells play a critical role in hepatitis B virus （HBV） pathogenesis. During acute, self-limited infections, these cells are instrumental to viral clearance; in chronic settings, they sustain repetitive cycles...CD8＋ T cells play a critical role in hepatitis B virus （HBV） pathogenesis. During acute, self-limited infections, these cells are instrumental to viral clearance; in chronic settings, they sustain repetitive cycles of hepatocellular necrosis that promote hepatocellular carcinoma development. Both CD8＋ T-cell defensive and destructive functions are mediated by antigen-experienced effector cells and depend on the ability of these cells to migrate to the liver, recognize hepatocellular antigens and perform effector functions. Understanding the signals that modulate the spatiotemporal dynamics of CD8＋ T cells in the liver, particularly in the context of antigen recognition, is therefore critical to gaining insight into the pathogenesis of acute and chronic HBV infection. Here, we highlight recent data on how effector CD8＋ T cells traffic within the liver, and we discuss the potential for novel imaging techniques to shed light on this important aspect of HBV pathogenesis.展开更多
基金supported by the Ministry of Science,ICT and Future Planning(MSIP)through the National Research Foundation of Korea(NRF)(RS-2024-00450201)supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute(KHIDI),funded by the Ministry of Health and Welfare,Republic of Korea(HI22C1374).
文摘We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo.To accomplish this,a conventional two-photon microscope was equipped with a 3D-printed holders,which stabilize the tissue surface within the focal plane of immersion objectives.Further computational image stabilization and noise reduction were applied,followed by superresolution radial fluctuations(SRRF)analysis,doubling image resolution,and enhancing signal-to-noise ratios for in vivo subcellular process investigation.Stabilization of<1μm was obtained by suction,and<25 nm were achieved by subsequent algorithmic image stabilization.A Mito-Dendra2 mouse model,expressing green fluorescent protein(GFP)in mitochondria,demonstrated the potential of long-term intravital subcellular imaging.In vivo mitochondrial fission and fusion,mitochondrial status migration,and the effects of alcohol consumption(modeled as an alcoholic liver disease)and berberine treatment on hepatocyte mitochondrial dynamics are directly observed intravitally.Suction-based stabilization in two-photon intravital imaging,coupled with computational super-resolution holds promise for advancing in vivo subcellular imaging studies.
文摘AIM： To investigate microvascular injury quantitatively in the small bowel with respect to cardiopulmonary bypass （CPB） and related mechanisms. METHODS： In 10 male SD rats, normothermic CPB was established and continued with a flow rate of 100-150 mL/kg per minute for 60 min, while another 10 sham-operated animals served as controls. An approximate 10-cm loop of the terminal ileum was exteriorized for observation by means of intravital fluorescence microscopy. The small bowel microcirculatory network including arterioles, capillaries, and collecting venules was observed prior to CPB, CPB 30 min, CPB 60 min, post-CPB 60 min and post-CPB 120 rain. The intestinal capillary perfusion, microvascular permeability and leukocyte adherence were also measured. RESULTS： The systemic hemodynamics remained stable throughout the experiment in both groups. In CPB animals, significant arteriolar vasoconstriction, blood velocity reduction and functional capillary density diminution were found. As concomitances, exaggerated albumin extravasation and increased leukocyte accumulation were also noted. These changes were more pronounced and there were no signs of restitution at the end of the observation period. CONCLUSION： CPB induces significant microcirculatory injury of the small bowel in rats. The major underlying mechanisms are blood flow redistribution and generalized inflammatory response associated with CPB.
文摘CD8＋ T cells play a critical role in hepatitis B virus （HBV） pathogenesis. During acute, self-limited infections, these cells are instrumental to viral clearance; in chronic settings, they sustain repetitive cycles of hepatocellular necrosis that promote hepatocellular carcinoma development. Both CD8＋ T-cell defensive and destructive functions are mediated by antigen-experienced effector cells and depend on the ability of these cells to migrate to the liver, recognize hepatocellular antigens and perform effector functions. Understanding the signals that modulate the spatiotemporal dynamics of CD8＋ T cells in the liver, particularly in the context of antigen recognition, is therefore critical to gaining insight into the pathogenesis of acute and chronic HBV infection. Here, we highlight recent data on how effector CD8＋ T cells traffic within the liver, and we discuss the potential for novel imaging techniques to shed light on this important aspect of HBV pathogenesis.
