Méthodes d'hyperrésolution optique et leurs applications pour l'imagerie biomédicale

Authors: Dehez, Harold
Advisor: Piché, MichelDe Koninck, Yves
Abstract: The nervous system cannot be analyzed without structural and functional observations of its hundred of billions of neurons. Since the discovery of fluorescent probes, at the end of the 20th century, optical microscopes became the best trade-off between structural (high resolution) and functional (live cells, high selectivity) analysis. However, it is well known, since the end of the 19th century, that the resolution of a focusing light microscope is limited by diffraction to about 200 nm; most of sub-cellular compartments cannot be discriminate with those modalities. We had to wait the end of the 90th, to witness the development of novel microscopy techniques "breaking the diffraction barrier"; STED (STimulated Emission Depletion), PALM (PhotoActivated Localization Microscopy) and STORM (STochastic Optical Reconstruction Microscopy) achieved spatial resolution of about 10 nm. However, most of those "superresolution" techniques are probe dependent (PALM/ STORM), or require high power laser and a complete modification of the microscope (STED). The goal of this thesis is to develop novel, low-power, and retrofittable "superresolution" procedures in laser scanning microscopy without limiting the specifications of conventional modalities (probe dependency, photodommage, simplicity, temporal resolution, . . . ). In this thesis, we propose to increase the resolution of laser scanning microscopes by structuring the illumination. The issue is tackled in two different ways: a direct approach where the excitation volume is reduced by changing the Gaussian beam used in conventional systems into a radially polarized transverse magnetic TM01 beam, and an indirect approach based on two successive illuminations of the sample with a Gaussian beam and a "dark" beam having a minimum of intensity at its center. With the later approach, we doubled the spatial resolution of confocal microscopes while keeping the specifications of the conventional systems except from doubling the temporal resolution.
Document Type: Thèse de doctorat
Issue Date: 2013
Open Access Date: 19 April 2018
Permalink: http://hdl.handle.net/20.500.11794/24493
Grantor: Université Laval
Collection:Thèses et mémoires

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