Critical Evaluation of FRET-Based Biosensor Performance: Implications for Measuring Ion Concentrations

Abstract

Fluorescent sensors are widely used to visualize, quantify, and reveal the dynamics of small molecules, secondary metabolites, metals, and ions. One of the great promises of such sensors is the ability to quantify cellular signals in precise locations with high temporal resolution, enabling the creation of real-time dynamic maps of cellular signals. Yet this is coupled with the challenge of how to ensure that sensors are measuring what we think they are measuring, developing robust approaches for quantification, and assessing whether sensors are perturbing the underlying biology. This talk will highlight our efforts to develop genetically encoded FRET-based sensors for quantitative mapping of zinc ions in cells. I will discuss approaches for defining whether sensors perturb cellular ions, the importance of carefully defining dynamic range, strategies to compare sensor functionality in vitro, in cells, and in organelles, and the specific challenges associated with quantifying ions in cellular organelles.