Monitoring Nanoscale Mobility of Small Molecules in Organized Brain Tissue with Time-Resolved Fluorescence Anisotropy Imaging

Abstract

Rapid movements of small ions and signaling molecules in the vicinity of central synapses determine how information is transferred by neural circuits of the brain. How fast such molecules can move in the crowded protein environment of live issue is, however, poorly understood. To enable the monitoring of molecular mobility on the nanoscale in situ, we have combined two-photon excitation microscopy and patch-clamp electrophysiology with time-resolved fluorescence anisotropy imaging (TRFAIM) in acute brain slices. TRFAIM exploits the fact that most fluorophores emit in the same polarization plane as that of excitation, with respect to the molecular structure. Because excitation and emission are separated by a nanosecond-range delay, the plane of emission diverges from that of excitation depending on the speed of molecular movements in space. We show that this methodology can be successfully adapted to two-dimensional mapping of diffusion rates inside and outside microscopic cellular compartments representing synaptic connections in the brain. This approach has a potential to unveil poorly understood determinants of diffusion-limited reactions and local molecular signal exchange underlying the functioning of central neural circuits.