Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

► We monitored the differential membrane behavior of glycine receptor α3 isoforms. ► Both ensemble average and single particle fluorimetric techniques were applied. ► Aggregation and diffusion were quantified using fixed and live-cell measurements. ► Evidence for molecular interaction of isoforms after membrane insertion is revealed. ► Considerations for fluorimetric probing of membrane protein behavior are reported.