利用單分子極化螢光相關顯微術研究脂質囊泡中的脂質動力學

Abstract

細胞是組成生命體的微小單位，在細胞之中有不同功能的膜狀組織以維持細胞的穩定，不同的組織有獨特的脂質組成比例以及偏好的曲度範圍；不僅如此，細胞彼此間也使用不同大小的脂質囊泡配合對應的運送方式，達到彼此間訊息和物質的交流。因此，研究脂質雙層以及脂質囊泡中的脂質動力學可以幫助人們更全面的了解生命的秘密。經過多年的發展，高解析度的單分子顯微術的出現賦予人們觀察複雜體環境中單分子行為的可能，也使得人們可以更進一步的了解生命的運作模式。在本篇研究中，我們架設出單分子極化螢光相關顯微鏡，並使用矽球輔助以合成出大小在一百奈米以下的人工囊泡，結合兩者，進行囊泡中脂質動力學的研究。我們在囊泡中參入單分子的染料分子（DiD），此染料分子可以和脂雙層結構造相容，當染料分子隨囊泡曲面運動時，其螢光行為也會隨位置而有所變化，收集其螢光訊號並以相關函數分析，我們可以得到染料行為的相關參數，例如:染料異構化的生命期、擴散係數以及三重態的生命期，這些參數透露出脂質堆疊狀況。我們合成出大小為一百奈米以下囊泡中，並觀察相關參數隨曲度增加的變化情況，推得曲度的增加使得脂質堆疊情況變得更緊密紮實而非稀疏；我們也在飽和脂質（DPPC）的狀況中，觀察到複合環境共存以及囊泡內外層的差異。就我們所知，這是第一次人們有一個直接方法可以觀察到如此微小囊泡中的脂質動力學，包含曲度效應以及複合環境間的差異，因此，我們認為此法可以使脂質動力學的研究有進一步的發展。此外，我們的方法僅僅使用單分子染料作為探測標的，這代表對樣品本身的干擾相當微小，故我們認為此方法非常有機會發展至活細胞的實驗，這將會是我們未來的發展方向。

Cells are tiny units which comprise different type living organism. There are different organs for different purposes in the cell and they are composed of lipid bilayers. In different organs, the lipid composition and curvature vary. In order to reach the balance of life, the concordant communication between cells is the essential issue for the life extension, no matter how the transport of nutrient or the transmission of signals are related to lipid vesicles. Therefore, the investigation of lipid dynamics in vesicles is the key to the complete understanding of life. In recent years, the development of single molecule microscopy with high resolution renders human the probability to visualize the single particle under complicated condition. In this research, we construct the single molecule fluorescence correlation microscope and synthesize the lipid vesicles with different size and phase to study the lipid dynamics in the vesicle. Fluorescence dye (DiD) is added in the lipid, so that we can track the fluorescence signal in the vesicle. By utilizing the correlation function for analysis, we may extract the lipid dynamics information such as isomerization life time, triplet decay life time and diffusion coefficient. These parameters help us gain insight into the environment of the vesicle. Given these parameters from different size and different lipid, we demonstrate the curvature effect and the co-existence of different phase domains in vesicles. When the curvature increases, the diffusion coefficient of lipid will decrease and the triplet state life time become longer, evidencing the packing condition of vesicle becomes tighter. In the case of DPPC, a common saturated lipid, we observe the co-existence of different environments in one vesicle, composed of solid phase and bouquet cluster. To our knowledge, it is the first time to observe the environmental difference in the vesicle with such small size. Our method is promising for cell investigation which can be anticipated in the future.