Development and application of fluorescent protein-based indicators for live cell imaging

BackgroundDevelopments in fluorescent molecular imaging techniques have enabled characterization of the localization and dynamics of particular molecules at the cellular and subcellular levels.HighlightAdvances in imaging devices (fluorescence microscopy) and fluorescent indicators have contributed to the development of fluorescence imaging techniques. In particular, cloning of green fluorescent protein from the bioluminescent jellyfish Aequorea victoria and the subsequent development of other fluorescent proteins have revolutionized imaging technologies and accelerated their application in molecular and cell biological studies. There are two types of genetically encoded fluorescent indicators, namely, fluorescence resonance energy transfer-based ratiometric indicators and single fluorescent protein (FP)-based intensiometric indicators. The principal advantages of FP-based indicators are their superior sensitivities and specificities for molecular and physiological events, which are achieved by the incorporation of naturally evolved protein sensor domains. Consequently, numerous fluorescent indicators that enable monitoring of intracellular signaling, enzyme activities, apoptosis, cell cycle progression, and other cellular events have been developed. FP-based indicators can be expressed in specific cells in a temporally controlled manner; hence, they are favorable for long-term in vitro and in vivo experiments.ConclusionThe advantages of FP-based indicators extend their application to understanding the functions of particular molecules and cellular events during physiological responses in live animals.