'CORRELATIVE FRET': NEW METHOD IMPROVES RIGOR AND REPRODICIBILITY IN DETERMINING DISTANCES WITHIN SYNAPTIC NANOSCALE ARCHITECTURE

摘要

A new correlative F?rster 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 photo-bleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100?, 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 70?, in contrast to the 62.5 ? using Alexa Fluor 594? IgG. Preliminary studies also show that using a FluoroNanogold?probe inhibits photo-bleaching. After one photo-bleaching session, Alexa Fluor 594? IgG fluorescence dropped to 19% of its original fluorescence; in contrast, after one photo-bleaching 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? IgG. 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.