Correlative FRET: New method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence; in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies.