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Nanoscale organization of proteins assemblies probed by polarized label free multiphoton fluorescence (Conference Presentation)
Author(s): Sophie Brasselet; Ellen Green; Peter Winlove
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Paper Abstract

Label free imaging is becoming of increasing interest for medical optics. Two- and Three-photon fluorescence, second harmonic generation (SHG), and coherent anti stokes Raman scattering (CARS) are key modalities for future in–situ diagnosis approaches in diseases such as cancer and neuropathologies. The addition of polarization monitoring provides moreover new elements of information on the way molecules are organized, making use of the coupling between rotating incident polarizations and oriented molecules. Extracting the dependence of nonlinear optical signals to polarization allows to image molecular order beyond the optical diffraction limit size. This parameter provides not only complementary fundamental understanding on molecular interactions, it is also a potential read-out for pathologies that inevitably perturb the architecture of protein assemblies. Whilst polarized nonlinear imaging has been demonstrated in SHG and CARS, it is less explored for two photon fluorescence (2PF) due to the lack of specificity of 2PF signals in biological tissues. In this work we show that label free polarized 2PF (P-2PF) is capable of revealing structure in dense protein fibres in vitro and intact tissues. We show in particular that this approach brings important insights into the origin of 2PF in beta-sheet like architectures such as in elastin or amyloids. We demonstrate moreover the sensitivity of elastin molecular order to the hydrophobicity of its environment and to mechanical forces. At last, we extend this approach to structures containing exogenous fluorescent labels, allowing us to explore the conformational properties of the constructs in cells.

Paper Details

Date Published: 4 March 2019
Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 1088222 (4 March 2019); doi: 10.1117/12.2509791
Show Author Affiliations
Sophie Brasselet, Institut Fresnel (France)
Ellen Green, Univ. of Exeter (United Kingdom)
Peter Winlove, Univ. of Exeter (United Kingdom)

Published in SPIE Proceedings Vol. 10882:
Multiphoton Microscopy in the Biomedical Sciences XIX
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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