Super-resolved imaging under total internal reflexion using random illumination microscopy (RIM)

Kevin Affannoukoué; Guillaume Maire; Thomas Mangeat; Simon Labouesse; Claire Estibal; Benoît Rogez; Guillaume Giroussens; Loic Legoff; Julien Savatier; Laurent Gallais; Marc Allain; Jérome Idier; Anne Sentenac

doi:10.1117/12.2624255

30 May 2022 Super-resolved imaging under total internal reflexion using random illumination microscopy (RIM)

Kevin Affannoukoué, Guillaume Maire, Thomas Mangeat, Simon Labouesse, Claire Estibal, Benoît Rogez, Guillaume Giroussens, Loic Legoff, Julien Savatier, Laurent Gallais, Marc Allain, Jérome Idier, Anne Sentenac

Author Affiliations +

Proceedings Volume PC12144, Biomedical Spectroscopy, Microscopy, and Imaging II; PC121440J (2022) https://doi.org/10.1117/12.2624255
Event: SPIE Photonics Europe, 2022, Strasbourg, France

Abstract

Total Internal Reflection Fluorescence Microscopy (TIRFM) exploits an evanescent field induced at the boundary between high and low refractive index media to selectively excite the sample inside a very thin region (from 100 to 300 nm depending on the illumination angle) above the coverslip surface. The minimum exposure of the sample to light above the excitation slice reduces significantly the out-of-focus fluorescence and phototoxicity which are major issues in live-cell imaging. It has become an indispensable tool in biology, in particular to study the molecular traffic at the cell plasma membranes. However, in many applications, the lateral resolution of TIRF, which is diffraction limited to about 300 nm, is not sufficient. In addition, the optical sectioning of the evanescent illumination of TIRF is seldom perfect. Propagative waves stemming from imperfections in the optical train of the instrument and/or light scattering by the sample itself are able to excite the fluorescence in the volume of the sample. When the latter is densely marked, these leaks result in out-of-focus fluorescence which deteriorates the signal to noise ratio. To improve simultaneously the lateral resolution and the image contrast, and to address the difficulties related to the control of the illumination patterns, we propose to adapt the recently developed Random Illumination Microscope (RIM) to the TIR configuration. We show that this approach yields a two-fold resolution gain and ameliorates the image contrast without compromising the ease of use of standard TIRFM. We apply TIRF-RIM to calibrated targets and to fixed and live biological samples with a sub-100nm resolution.

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Kevin Affannoukoué, Guillaume Maire, Thomas Mangeat, Simon Labouesse, Claire Estibal, Benoît Rogez, Guillaume Giroussens, Loic Legoff, Julien Savatier, Laurent Gallais, Marc Allain, Jérome Idier, and Anne Sentenac "Super-resolved imaging under total internal reflexion using random illumination microscopy (RIM)", Proc. SPIE PC12144, Biomedical Spectroscopy, Microscopy, and Imaging II, PC121440J (30 May 2022); https://doi.org/10.1117/12.2624255

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KEYWORDS

Microscopy

Reflection

Image resolution

Luminescence

Calibration

Microscopes

Scattering

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