Strategies for Multiplexed Biosensor Imaging to Study Intracellular Signaling Networks

Signal transduction processes are a necessary component of multicellular life, and their dysregulation is the basis for a host of syndromes and diseases. Thus, it is imperative that we discover the complex details of how signal transduction processes result in specific cellular outcomes. One of the primary mechanisms of regulation over signaling pathways is through spatiotemporal control. However, traditional methods are limited in their ability to reveal such details. To overcome these limitations, researchers have developed a variety of genetically encodable, fluorescent protein-based biosensors to study these dynamic processes in real time in living cells. Due to the complexities and interconnectedness of signaling pathways, it is thus desirable to use multiple biosensors in individual cells to better elucidate the relationships between signaling pathways. However, multiplexed imaging with such biosensors has been historically difficult. Nevertheless, recent developments in designs and multiplexing strategies have led to vast improvements in our capabilities. In this review, we provide perspectives on the recently developed biosensor designs and multiplexing strategies that are available for multiplexed imaging of signal transduction pathways.