Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera
Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera
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DOI：
10.1021/ac0624546
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发表时间：
2007-06-15
影响因子：
7.4
通讯作者：
Wohland, Thorsten
中科院分区：
文献类型：
--
作者：
Kannan, Balakrishnan;Guo, Lin;Wohland, Thorsten
利用电子倍增电荷耦合器件（EMCCD）相机构建了空间分辨全反射荧光相关显微镜（TIR-FCM）系统。该系统用于测定平面脂质双分子层中脂质分子的扩散系数，以及脂质和表皮生长因子受体（EGFR）在中国仓鼠卵巢（CHO）细胞膜上的表达。对相邻像素之间“串扰”的评估表明，可以实现比以前提出的更高程度的复用[Kannan， B. et al.]。分析的。化学，2006,78,3444-51]。对于CCD中包含20条线的感兴趣区域，该系统的最佳时间分辨率为4 ms，足以确定脂质双分子层和活细胞中膜蛋白的膜扩散。在这项工作中，使用TIR-FCM设置，以4.8 ms的时间分辨率同时测量了1600个自相关函数。该区域对应于一个40 x 40像素的感兴趣区域，尺寸为11.3 x 11.3 μ m(2)，足够大，可以同时测量整个细胞的下膜。
A spatially resolved total internal reflection fluorescence correlation microscopy (TIR-FCM) system is constructed with an electron multiplying charge-coupled device (EMCCD) camera. The system was used to determine diffusion coefficients of lipid molecules in a planar lipid bilayer, and lipids and epidermal growth factor receptor (EGFR) proteins on cell membranes of Chinese Hamster Ovary (CHO) cells. The evaluation of the "cross talk" between neighboring pixels suggests that a higher degree of multiplexing can be achieved than was previously proposed [Kannan, B. et al. Anal. Chem. 2006, 78, 3444-51] using the same camera with a focused laser excitation. The best time resolution possible with this system is 4 ms for a region of interest comprising 20 lines in the CCD and is good enough to determine membrane diffusion in lipid bilayers and of membrane proteins in living cells. In this work, using a TIR-FCM setup, 1600 autocorrelation functions were measured simultaneously with a time resolution of 4.8 ms. This area corresponds to a 40 x 40 pixel region of interest with a dimension of 11.3 x 11.3 mu m(2) and is sufficiently large to allow the measurement of the lower membrane of a whole cell simultaneously.