Frequency-domain-based FRET-FLIM microscopy

COS-7 cells were co-transfected with carboxyl terminally tagged GluN1-GFP together with HA-GluN2A and with the carboxyl terminally tagged D1R (WT or S397D)-mCherry in a proportion of 1:1:1, unless stated otherwise. mCherry alone was used as a FRET-negative control. Cells were imaged with an HCX PL Apo 63x oil NA 1.4 objective using an appropriate GFP filter set. Cells were excited using a sinusoidally modulated 3 W 478 nm LED (light-emitting diode) at 36 MHz under wild-field illumination. Emission was collected using an intensified CCD LI2CAM camera (Lambert Instrument BV, Groningen, The Netherlands). Lifetimes were calibrated using a solution of erythrosin B that was set at 0.086 ns. The lifetime of the sample is determined from the fluorescence phase-shift between the sample and the reference from a set of 12 phase settings using the manufacturer’s LI-FLIM software.