ABSTRACT
Microglia is a population of myeloid resident cells in the central nervous system (CNS). This chapter describes and exemplifies image-processing implementations for single-chain ratiometric Foster resonance energy transfer (FRET) biosensors to study microglial function. It shows that such ratiometric FRET approach is extremely sensitive for routine inspection of signalling events in real time with high spatio-temporal resolution in living microglia. The chapter exemplifies the use of this ratiometric FRET approach in the subcellular activation of the tyrosine kinase c-Src in live microglia exposed to LPS, a component of bacterial cell wall classically known to trigger a proinflammatory response on these cells. Understanding the kinetics of c-Src activation in different microglial subcellular domains may help explain microglia-associated neuronal impairment during chronic neurodegeneration. The routines for the c-Src ratiometric FRET biosensor can be perfectly accommodated for other FRET-based ratiometric nanosensors with only minimal adjustments.
