Real-time optical imaging of the liver: diagnosis and prediction of treatment response

Multiphoton microscopy (MPM) has become increasingly popular and widely used in both basic and clinical liver studies over the past few years. The fluorescence lifetime imaging (FLIM) can be coupled with MPM to add additional quantitation and specificity to the detection of endogenous fluorescent species in the liver as well as exogenous molecules, nanoparticles and therapeutic cells that applied to the liver both in vitro and in vivo. These technologies provide deep penetration of live tissues with less photobleaching and phototoxicity, and help our better understanding of the cellular morphology, microenvironment, immune responses and spatiotemporal dynamics of drugs and therapeutic cells in the healthy and diseased liver.Taking advantages of MPM-FLIM, the aim of this project is to develop novel optical imaging methods for diagnosis and prediction of treatment response in liver diseases. The main achievements obtained in this thesis are listed as below:(1) Conventional histology with light microscopy is essential in the diagnosis of most liver diseases. Recently, a concept of real-time histology with optical biopsy has been advocated. Live mice livers (normal, with fibrosis, steatosis, hepatocellular carcinoma and ischemia-reperfusion injury) were imaged by MPM-FLIM for stain-free real-time histology. The acquired MPM-FLIM images were compared with conventional histological images. MPM-FLIM imaged subsurface cellular and subcellular histopathological hallmarks of live liver in mice models at high resolution. Additional information such as distribution of stellate cell associated autofluorescence and fluorescence lifetime changes was also gathered by MPM-FLIM simultaneously, which cannot be obtained from conventional histology. MPM-FLIM could visualise the cell morphology and microenvironment changes in diseased livers without conventional biopsy or administration of fluorescent dyes.(2) Oxidative stress reflects an imbalance between reactive oxygen species (ROS) and antioxidants, which has been reported as an early unifying event in the development and progression of various diseases and as a direct and mechanistic indicator of treatment response. A transition-metal complex-based sensing platform was developed for in vivo MPM-FLIM imaging of oxidative stress at a single cell resolution. By combining fluorescence intensity and fluorescence lifetime imaging, this sensing platform accurately localised ROS and glutathione (GSH) within the liver, and quantified their changes during liver injury. This precedes changes in conventional biochemical and histological assessments. Importantly, the optical oxidative stress index (OSI), expressed as the fluorescence intensity ratio of ROS and GSH probes, has significant implications for sensitive, spatially configured and quantitative assessment of metabolic status and drug response, which could reveal mechanistic insights and accelerate drug development studies.(3) Although mesenchymal stem cells (MSCs) present a promising tool in cell therapy for the treatment of liver cirrhosis, the in vivo distribution of administered MSCs has still been poorly understood, which hampers the precise prediction and evaluation of their therapeutic efficacy. The spatiotemporal disposition of administered MSCs in the liver was directly visualized using MPM and the cell quantity was assessed using flow cytometry. A physiologically based kinetic model was developed to characterise the in vivo distribution of MSCs. This model was further validated with multiple external datasets, indicating potential inter-route and inter-species predictive capability. The results suggest that the targeting efficiency of MSCs is determined by the lung retention and interaction between MSCs and target organs, including cell arrest, depletion and release. By adapting specific parameters, this model can be easily applied to abnormal conditions or other types of circulating cells for designing treatment protocols and guiding future experiments.