Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

79-POS Board 40 Supercritical Angle Microscopy: Surface-Sensitive Nanoscale Fluorescence (SAF) and Raman Microscopy (SAR) for Imaging of Surfaces and Cells Stefan Seeger , Diana Serrano.Valentin Dubois. University of Zurich, Zurich, Switzerland. Surface sensitive detection technologies are of great interest to study the interaction of biomolecules with surfaces, in particular. Some time ago, we have introduced Supercritical Angle Fluorescence Microscopy and applied it to study the realtime observation of biomolecules with lipid bilayers, in particular proteins involved in neurodegenerative diseases /1-4/. The simultaneous observation of light collected above and below the critical angle allows even the localization of the emitter down to a few nanometer (<10nm)/5/. Meanwhile, this technique has been combined with other superresolution techniques /6/. Now, we extend this technology to the Raman spectroscopic regime /7/. Raman spectroscopy is a non-invasive technique able to provide chemical and compositional information about a variety of materials and molecular entities. We show for the first time that the collection of Raman scattered signals exceeding the critical angle of total internal reflection leads to surface-confined nanometre axial resolution. This high axial selectivity gives access to the intrinsic fingerprint of surface-related molecular specimens and can provide evidence about their distinguished structural and functional features. The richness of the spectroscopic information obtained through the supercritical angle Raman (SAR) collection path is demonstrated by simultaneously comparing its output with that of a classical confocal collection path. Furthermore, the proposed SAR technique is a versatile microscopy approach which can be used alone or in combination with amplified Raman modalities such as SERS. /1/ Ruckstuhl T, Rankl M, Seeger S: Highly sensitive biosensing using a Supercritical Angle Fluorescence (SAF) instrument, Biosensors &Bioelectronics 18 (9) 1193-1199 (2003) /2/ Ruckstuhl T., Verdes D., Winterflood C., Seeger S.: Simultaneous near-field and far-field fluorescence microscopy of single molecules, Optics Express 19, 6836-6844 (2011) /3/ Reynolds N., Soragni A., Rabe M., Verdes D., Liverani E., Handschin S., Riek R., Seeger S.: Mechanism of membrane interaction and disruption by α-Synuclein, Journal of the American Chemical Society 133, 19366-19375 (2011) /4/ Rabe M., Soragni A., Reynolds N., Verdes D., Liverani E., Riek R., Seeger S.: On-surface aggregation of α- Synuclein at nanomolar concentrations, results in two distinct growth mechanisms, ACS Chemical Neuroscience 4, 408-417 (2013) /5/ Winterflood C., Ruckstuhl T., Verdes D., Seeger S.: Nanometer Axial Resolution by Three-Dimensional Supercritical Angle Fluorescence Microscopy (3D-SAFM), Physical Review Letters 105, 108103 (2010) /6/ Bourg N, Mayet C, Dupuis G, Barroca T, Bon P, Lécart S, Fort E, Lévêque-Fort S: Direct optical nanoscopy with axially localized detection, Nature Photonics 9, 587–593 (2015) /7/ Serrano D, Seeger S: Supercritical angle Raman microscopy: a surface-sensitive nanoscale technique without field enhancement, in press 2017

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