Quantitative imaging with fluorescent biosensors.

Molecular activities are highly dynamic and can occur locally in subcellular domains or compartments. Neighboring cells in the same tissue can exist in different states. Therefore, quantitative information on the cellular and subcellular dynamics of ions, signaling molecules, and metabolites is critical for functional understanding of organisms. Mass spectrometry is generally used for monitoring ions and metabolites; however, its temporal and spatial resolution are limited. Fluorescent proteins have revolutionized many areas of biology-e.g., fluorescent proteins can report on gene expression or protein localization in real time-yet promoter-based reporters are often slow to report physiologically relevant changes such as calcium oscillations. Therefore, novel tools are required that can be deployed in specific cells and targeted to subcellular compartments in order to quantify target molecule dynamics directly. We require tools that can measure enzyme activities, protein dynamics, and biophysical processes (e.g., membrane potential or molecular tension) with subcellular resolution. Today, we have an extensive suite of tools at our disposal to address these challenges, including translocation sensors, fluorescence-intensity sensors, and Förster resonance energy transfer sensors. This review summarizes sensor design principles, provides a database of sensors for more than 70 different analytes/processes, and gives examples of applications in quantitative live cell imaging.