In Vivo Fluorescence/Photoacoustic Dual-Modality Labeling And Long-Term Imaging Of Liver Resident Immune Cells

In recent years the liver has been recognized as an important regional immune organ with a unique blood sinusoidal structure and maintenance of body homeostasis,which is closely associated with the development and progression of disease.The intrinsic immune system,as the innate defense system of the organism,plays a rather important role in the defense of the organism against the invasion of exogenous pathogens.KCs(Kupffer cells)are macrophages that develop from the yolk sac and settle in the liver,accounting for about 80%-90% of the total number of intrinsic macrophages.Although KCs have been known for a long time,their distribution pattern in the liver lobules and their phagocytic degradation ability are not well understood,and it is also unclear how KCs and other immune cells are dynamically involved in the development of liver diseases.Fluorescence imaging of the liver using antibodies as tracers of KCs is usually difficult to balance the imaging depth,field of view and spatial resolution,and cannot simultaneously obtain fine structural information of the liver lobules,the basic structural unit of the liver,and accurate localization and functional information of KCs.Photoacoustic imaging can compensate for the shortcomings of fluorescence confocal imaging in terms of imaging depth,field of view and vascular imaging,however,the current lack of bimodal imaging marker probes for liver immune cells makes it difficult to effectively observe liver immune structures and immune cell interactions in in vivo imaging.Intravital imaging of the abdomen with high spatiotemporal resolution often encounters a significant challenge: the irregular movement caused by respiratory fluctuation and heartbeat severely affects the stability of the focal plane of optical microscopy,resulting in unclear images.It is difficult to effectively observe the physiological structure and cell movement patterns of the liver.The ideal imaging window for the liver should slow down the jitter of the liver and eliminate the artifacts produced without changing the physiological state of the liver.Establishing an in vivo long-time,large-field,high-quality imaging method suitable for abdominal organs is essential to obtain the fine hepatic blood sinusoidal structure and the location distribution of immune cells as well as the motility characteristics of the liver.Based on these problems,this dissertation developed a bimodal fluorescent/optoacoustic nanoprobe that can specifically target liver KCs,invented a drawer-type abdominal window model that can be used for bimodal imaging,and applied fluorescent/optoacoustic bimodal imaging to investigate the normal liver physiological state and liver metastasis model in depth.The main findings are as follows.(1)Pomegranate-like structured nanoparticles that can be rapidly self-assembled were invented,and hydrophobic near-infrared fluorescent dyes can be efficiently self-assembled into pomegranate seeds(4~5nm),tens of thousands of which form a pomegranate nanoparticle with a particle size of 399 nm.Due to the unique optical properties of the pomegranate nanoparticles and their particle size of hundreds of nanometers,they are rapidly taken up by KCs in the liver after intravenous injection,with an uptake rate of98.8%.As a result,the phagocytic ability of KCs can be reflected by detecting the photoacoustic signal in the liver by photoacoustic imaging,and the degradation function of KCs can be reflected by detecting the fluorescence signal in the liver.(2)Photoacoustic imaging of in vivo liver clearly demonstrates the fine structure of longitudinally and horizontally arranged liver lobules and hepatic blood sinusoids.Dual-wavelength photoacoustic imaging(523 nm and 744 nm)results showed a linear relationship(R2 = 0.751)between the density of KCs within individual liver lobules and their distance from the central vein(rn/rmax);characterized the relationship between the area size of KCs and their spatial location,which was linearly correlated with the spatial location of KCs(rn/rmax)(R2 = 0.7209),with an increase in area as the rn/rmax ratio increased along the CV-PT(Central Vein-Portal Vein)axis;the phagocytic capacity of KCs varied along the CV-PT axis of the liver lobules,with a sawtooth fluctuation between rn/rmax ratios of 0.167 and 0.3(close to CV),with the lowest and highest values occurring at rn/rmax ratios of 0.2 and 0.233;in vivo Fluorescence imaging of liver showed that KCs slowly released fluorescent signals after phagocytosis of nanoparticles;comparison with commercial latex particles showed a clearer morphology of KCs.(3)A drawer-type abdominal window with acrylic/resin as the imaging medium was developed and applied to long-duration in vivo fluorescence/photoacoustic imaging of the liver,and the imaging duration can exceed 10 days.The imaging artifacts are eliminated without altering the physiological structure and state of the liver.By performing long-duration fluorescence/photoacoustic imaging of trans-splenial liver metastases of tf RFP-B16 tumors in a drawer-window model of CXCR6-GFP mice,we obtained image information of hepatic blood sinusoids and immune cells during tumor development.The results showed that the density of Natural Killer T cells(NKT)was 11-fold and 10.1-fold higher in the tumor parenchyma than in the extra-tumor and control areas,indicating that the distribution of NKT cells in the liver metastasis microenvironment had prominent spatial characteristics,with most of them clustered in and around the parenchyma of the tumor metastases.KCs within the tumor parenchyma had a smaller cell area and their morphological characteristics were the same as those in the peri-tumor area,tending to be contracted and amoeboid.The KCs in the extra-tumor area had a larger cell area and morphological characteristics similar to those of the control KCs,tending to be extended,spindle-shaped,and angular.In summary,this project successfully solved the challenge of efficient target labeling of KCs with simultaneous photoacoustic/fluorescence dual-modality imaging,revealing the strategic arrangement and function of KCs in the living liver;the new method established for microscopic imaging of the living liver provides an effective tool for in vivo long-duration high-resolution visualization of changes in vascular structure and immune cells during liver disease.