利用多光子螢光光譜、顯微術以及螢光生命週期顯微術來診斷肝癌

Abstract

藉由多光子螢光光譜、多光子非線性顯微術和螢光生命週期影像，我們成功建立了一個膽紅素分子影像的平台，觀察在肝癌 (hepatocellular carcinoma)中不正常的膽紅素代謝。利用紅外光的鉻貴橄欖石飛秒雷射做為光源，能避免激發其他生物體內螢光分子的雙光子螢光，而讓膽紅素的紅螢光成為多光子螢光影像的主要對比。藉由膽紅素的螢光對比，可半定量的檢測出膽紅素的分布和數量。 我們發現，在非腫瘤的肝組織區域，膽紅素有很強的自發螢光，這些螢光分佈在肝細胞內的顆粒狀囊泡。而在分化不全的肝癌細胞中，這些發螢光的顆粒狀囊泡都消失，造成多光子螢光強度明顯的減弱。如果發現有膽汁淤積的切片，這代表是分化比較好的肝癌細胞，跟非腫瘤部位的切片比較起來擁有較強的螢光強度並多出一個800 nm的螢光峰。利用多光子的螢光光譜、螢光強度和螢光生命週期，膽紅素是可用來檢測肝癌的分子標誌。我們可以靈敏的檢測肝癌中不正常的膽紅素代謝而不需要額外的化學標記。

With multi-photon fluorescence spectra, multi-photon nonlinear microscopy and FLIM images, we successfully build up a bilirubin molecular imaging platform and imaged the metabolic disorders of bilirubins in HCC. By using an infrared femtosecond Cr:forsterite laser source with its wavelength away from two-photon resonance of most endogenous pigments, the distribution and amount of bilirubins can be semi-quantitively mapped over low-background autofluorescences. For non-tumor regions of liver tissues, the autofluorescences of bilirubins are strong and emitted from granular vesicles in hepatocytes. In poorly-differentiated HCC, the granular vesicles which emit fluorescence disappear and the intensity of multi-photon fluorescence is apparently reduced. If the hepatocytes have cholestasis, which is the signature of well-differentiated HCC, the fluorescence intensity were much higher than non-tumor part and the spectra showed an extra 800nm peak. These results indicate that, based on the multiphoton microscopy of autofluorescence spectra, intensities, and lifetimes, endogenous bilirubins are useful markers to indicate the presence and the staging of HCC. Without an extra labeling or chemical assays, disorder of bilirubins metabolisms in HCC can be sensitively detected.