Total Internal Reflection Fluorescence Microscopy for Single-molecule Imaging in Living Cells

Cell Structure and Function

Yasushi Sako¹  Takeshi Uyemura² 

[1] Department of Physiology and Biosignaling, Graduate School of Medicine, Osaka University;Department of Systems and Human Science, Graduate School of Engineering Science, Osaka University

关键词: evanescent field microscopy;    nanobiology;    nanotechnology;    protein dynamics;    single-molecule kinetics;   

DOI  :  10.1247/csf.27.357

学科分类：分子生物学，细胞生物学和基因

来源: Japan Society for Cell Biology

【 摘 要 】

References(33)Cited-By(31)Marvelous background rejection in total internal reflection fluorescence microscopy (TIR-FM) has made it possible to visualize single-fluorophores in living cells. Cell signaling proteins including peptide hormones, membrane receptors, small G proteins, cytoplasmic kinases as well as small signaling compounds have been conjugated with single chemical fluorophore or tagged with green fluorescent proteins and visualized in living cells. In this review, the reasons why single-molecule analysis is essential for studies of intracellular protein systems such as cell signaling system are discussed, the instrumentation of TIR-FM for single-molecule imaging in living cells is explained, and how single molecule visualization has been used in cell biology is illustrated by way of two examples: signaling of epidermal growth factor in mammalian cells and chemotaxis of Dictyostelium amoeba along a cAMP gradient. Single-molecule analysis is an ideal method to quantify the parameters of reaction dynamics and kinetics of unitary processes within intracellular protein systems. Knowledge of these parameters is crucial for the understanding of the molecular mechanisms underlying intracellular events, thus single-molecule imaging in living cells will be one of the major technologies in cellular nanobiology.

【 授权许可】

Unknown