Zenisek David, Perrais David
CSH Protoc. 2007 Oct 1;2007:pdb.prot4863. doi: 10.1101/pdb.prot4863.
INTRODUCTIONAlthough electrophysiological techniques such as membrane capacitance measurements, electrochemical detection, and post-synaptic recordings are powerful ways of studying exocytosis, information concerning any steps prior to vesicle fusion must be inferred indirectly. Total internal reflection fluorescence microscopy (TIRFM) is a powerful technique for studying events that, like exocytosis, occur near a cell surface. The technique allows selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass. In this protocol, TIRFM is used to investigate the steps leading up to vesicle fusion in both retinal bipolar neurons and chromaffin cells by directly imaging synaptic vesicles and dense core granules prior to and including exocytosis.
引言
尽管诸如膜电容测量、电化学检测和突触后记录等电生理技术是研究胞吐作用的有力方法，但关于囊泡融合之前任何步骤的信息都必须间接推断。全内反射荧光显微镜（TIRFM）是一种用于研究像胞吐作用一样发生在细胞表面附近事件的强大技术。该技术允许对最接近诸如玻璃等高折射率物质的荧光分子进行选择性成像。在本实验方案中，通过在胞吐作用之前及包括胞吐作用期间直接对突触囊泡和致密核心颗粒进行成像，TIRFM被用于研究视网膜双极神经元和嗜铬细胞中囊泡融合之前的步骤。
