「同調反史托克拉曼散射顯微技術」應用於脂肪肝組織成像

Abstract

脂肪肝是一種肝臟內部累積過多脂肪微粒的病症，嚴重時會引起肝臟細胞發炎，導致肝臟纖維化甚至肝硬化。目前常見的醫學影像工具 (如腹部超音波檢查、電腦斷層掃描攝影、核磁共振掃描攝影…等)，由於訊號無化學專一性，大多只能判別脂肪肝存在與否或提供粗略的半定量分析。此外，以上醫學影像的空間解析度僅及微米尺度，對於需要細胞層級成像的基礎研究也稍嫌不足。因此，在許多情況仍須仰賴侵入式且耗時的組織切片配合顯微鏡檢驗。例如，肝臟移植手術中為評估肝臟的脂肪浸潤程度，減少移植手術的風險，便經常利用肝臟穿刺切片檢查。本研究中，我們利用自行架設的「同調反史托克拉曼散射」顯微系統 (Coherent anti-Stokes Raman scattering microscopy, CARS microscopy)，對脂肪肝老鼠的肝臟組織進行高解析度化學成像。利用CARS訊號對化學官能基具有專一性的特性，我們成功得到顯示肝組織內脂肪微粒分布的CARS影像，並觀測到老鼠在不同階段的脂肪肝病程時，肝組織內脂肪微粒大小及分布的變化。拉曼光譜鑑定顯示這些脂肪微粒之主要成分為三酸甘油酯，與生化分析結果相符。利用自行開發的影像分析方法，可進一步得到肝組織內的脂肪含量。所得結果與生化分析作比較，發現兩者之間有良好的線性相關性。此方法的最大優點在於不須對樣品進行切片或染色，可即時顯示組織內脂肪微粒分佈，並得到肝臟內脂肪濃度的資訊。我的研究結果顯示CARS顯微技術可對脂肪肝做即時及定量的檢測，未來有潛力發展成為全新的手術中 (intra-operative) 醫學影像檢測工具。

Fatty liver, which is considered the most prevalent liver disease may progress from benign fatty liver, steatohepatitis, cirrhosis, to hepatocellular carcinoma. In light of its high prevalence and severe outcome, it is essential to evaluate hepatic fatty change. Although non-invasive medical imaging tools such as ultrasound are commonly employed, invasive liver biopsy followed by histological examination under optical microscopes is still indispensable for the diagnosis of fatty liver. Nevertheless, histological examination requires extensive preparation procedures thereby very time consuming. In this thesis, I report the development of a coherent anti-Stokes Raman scattering (CARS) microscope and demonstrate label-free, real-time, three-dimensional, high-resolution, fat-specific pathological imaging of liver tissues on an animal model of fatty liver. By using CARS microscopy, detailed cellular morphology and intracellular fat droplets have been clearly resolved on untreated liver tissues. The progression of the hepatic fatty change has also been quantitatively assessed in a time-coursed study. Comparison of the result obtained by CARS imaging and that by parallel biochemical analysis exhibits an excellent linear correlation (n= 12, R = 0.89) on a pathologically significant range (from 15 to 190 mg of fat per g tissue). This finding suggests that CARS microscopy possesses not only great sensitivity to diagnose the onset of the fatty change in liver but also a sufficient dynamic range to monitor the severity of fatty liver diseases. Our results show that CARS microscopy has a potential to become a powerful clinical tool not only for in situ intraoperative imaging of liver but also for longitudinal studies aiming to evaluate the natural course of hepatic steatosis.