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Correlative FRET: new method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture
Author(s): Heather Shinogle-Decker; Noraida Martinez-Rivera; John O'Brien; Richard D. Powell; Vishwas N. Joshi; Samuel Connell; Eduardo Rosa-Molinar
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Paper Abstract

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.

Paper Details

Date Published: 23 February 2018
PDF: 8 pages
Proc. SPIE 10499, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV, 1049920 (23 February 2018); doi: 10.1117/12.2290806
Show Author Affiliations
Heather Shinogle-Decker, The Univ. of Kansas (United States)
Noraida Martinez-Rivera, The Univ. of Kansas (United States)
John O'Brien, The Univ. of Texas Health Science Ctr. at Houston (United States)
Richard D. Powell, Nanoprobes, Inc. (United States)
Vishwas N. Joshi, Anvesha Labs. (United States)
Samuel Connell, Intelligent Imaging Innovations, Inc. (United States)
Eduardo Rosa-Molinar, The Univ. of Kansas (United States)

Published in SPIE Proceedings Vol. 10499:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV
Thomas G. Brown; Carol J. Cogswell; Tony Wilson, Editor(s)

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