Analysis of TrkB Receptor Activity Using FRET Sensors
Here, we describe the use of 2-photon fluorescence lifetime imaging (2pFLIM) of a Förster resonance energy transfer (FRET) sensor to study the spatial and temporal activity pattern of the BDNF receptor TrkB. Combining 2pFLIM with laser uncaging of MNI-glutamate over single dendritic spines in organotypic hippocampal slices allows measurement of glutamate-induced changes in TrkB activity within a single spine or dendrite. This protocol covers the installation and setup of requisite hardware and software, the use of the software to acquire data, and the analysis of the data. A prerequisite for attempting this protocol is proficiency with 2-photon imaging of live cells and a microscope that is controlled by ScanImage 3.8 (Vidrio Technologies).
Keywords2-Photon Dendritic spines FLIM FRET Glutamate uncaging
This work was supported by NS05621 (NIH) to JOM.