利用多光子顯微術量化活體內肝細胞分子代謝

Abstract

雙光子螢光顯微術自西元1990年以來被廣泛應用活體影像的研究上。利用其非侵入性和光學切片的特性，搭配活體肝臟觀測平台，可得到高影像品質的肝臟動態資訊。為了探討肝臟的基礎生物物理，嘗試定量活體內肝細胞代謝的分子數量，在得到肝臟代謝趨勢的同時，還能提供肝細胞分子數量的實際改變。 以6-carboxyfluoescein diacetate為試劑，藉由觀測小鼠肝細胞將6-carboxyfluorescein diacetate酯化成6-carboxyfluorescein並排出細胞的過程，將肝細胞內6-carboxyfluorescein的螢光訊號針對肝臟組織與球面相差造成的訊號衰減進行校正後，與已知濃度的6-carboxyfluorescein螢光劑進行訊號強度比較，推得肝臟代謝期間肝細胞內6-carboxyfluorescein的最大分子濃度約在10~100μM，並乘上估計的肝細胞體積5000μm3，得到肝臟代謝期間內一個肝細胞代謝的最大6-carboxyfluorescein分子數量約在107到108個左右。

Two photon fluorescence microscopy has been extensively employed on in vivo imaging since A.D.1990. It is possible to get the high quality dynamic imaging of liver by combining its non-invasive property and optical sectioning ability with liver chamber. In order to investigate fundamental biophysics of liver, try to quantify molecule metabolism of hepatocyte to offer the actual change of the molecule quantity of hepatocyte while getting the trend of hepatic metabolism. Using 6-carboxyfluorescein diacetate as reagent, observe the process of 6-carboxyfluorescein diacetate esterifies into 6-carboxyfluorescein in mouse hepatocyte. Correcting the signal attenuation of 6-carboxyfluorescein in hepatocyte induced by absorption and scattering of liver tissue and spherical aberration caused of refractive index mismatch, compare it with fluorescent intensity of known concentration of 6-carboxyfluorescein to get the maximum molecule concentration in hepatocyte is 10~100μM during hepatic metabolism. The maximum molecular number of 6-carboxyfluorescein in each hepatocyte is 107 to 108 by multiplying the volume of hepatocyte 5000μm3 to the concentration.