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