Visualising nanoscale interactions across the mesoscale through FRET imaging

Fluorescence resonance energy transfer (FRET) imaging is a technique commonly used in biomolecular research to visualise molecular proximity. This occurs when two molecules are sufficiently near to each other to non-radiatively transfer the energy from the donor molecule and excite fluorescence from an acceptor molecule [1]. While this provides essential information about interactions occurring on a molecular level, the small fields-of-view of FRET [2] restrict the information obtained to a small number of cells, and does not give a clear understanding of how these interactions occur across an entire cell population. Here, we present the possibility of using FRET in conjunction with confocal Mesolens [3] imaging as a means of assessing molecular proximity across an extended field of view of up to 5.0 mm x 5.0 mm. We show that it is possible to image FRET interactions occurring through sensitised emission with the Mesolens, with the presence of FRET confirmed using known FRET standards with varying linker lengths. We propose this method for the study of interactions and structural organisation beyond the nanometre range, bridging molecular detail with cellular architecture in large cell populations. 1. T. Förster, “Energy migration and fluorescence,” J. Biomed. Opt. 17(1), 011002 (2012) [doi:10.1117/1.JBO.17.1.011002]. 2. D. R. Matthews et al., “Time‐lapse FRET microscopy using fluorescence anisotropy,” Journal of Microscopy 237(1), 51–62 (2010) [doi:10.1111/j.1365-2818.2009.03301.x]. 3. G. McConnell et al., “A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout,” eLife 5, e18659 (2016) [doi:10.7554/eLife.18659].