Assia Benachir will defend her Thesis entitled “Methods and instrumentation for single and two-photon fluorescence random illumination microscopy” on Tuesday, December 16th at 10:00 a.m. in Ponte Amphitheater, campus St Jérôme in Marseille.
The presentation and the slides will be in English.
The Jury members are :
– Randy Bartels, Morgridge Institute of Research, University of Wisconsin-Madison, President
– Ignacio Izeddin, Institut Langevin, ESPCI Paris, Examinator
– Sandrine Lévêque-Fort, ISMO, Université Paris-Saclay, Rapporteure
– Hilton Barbosa de Aguiar, Laboratoire Kastler-Brossel, ENS Paris, Rapporteur
– Hervé Rigneault, Institut Fresnel, Thesis Director
– Sandro Heuke, Institut Fresnel, Thesis Co-director
Abstract : Fluorescence microscopy is an essential tool for biological and medical imaging applications, owing to the wide choice in fluorescent labels, their high specificity, and sensitivity. Widefield imaging, where the entire field of view is illuminated at once, benefits from its technical simplicity and high acquisition speed. However, it lacks optical sectioning, i.e. the capability of distinguishing between planes inside a three-dimensional sample. Point-scanning approaches, particularly two-photon fluorescence, enable optical sectioning, but at a cost. Indeed, increasing the acquisi-tion speed while retaining a high signal to noise ratio requires higher excitation laser power, which in turn increases the risks of photodamage and photobleaching. In this PhD work, we propose novel experimental approaches for single and two-photon fluorescence widefield optical sectioning microscopy. Using random speckle illumination, we apply two widefield techniques, Dynamic Speckle Illumination (DSI) and Random Illumination Microscopy (RIM), to achieve optical sectioning down to 2 µm, over large fields of view of up to 250 µm. In addition to that, the RIM super-resolution algorithm enables a two-fold lateral resolution improvement, while keeping the experimental setup straightforward and robust to misalignment. First, we show that a zwitterion-doped nematic liquid crystal diffuser allows for precise and highly tunable control of the speckle decorrelation speed. Matching the speckle speed to the exposure time of the camera, we are able to demonstrate widefield fluorescence DSI imaging, with an imaging rate up to 14 Hz. In a second project, we make use of two-photon excitation to achieve deeper pen-etration into biological tissues. We also demonstrate multicolour widefield imaging over a large field of view using two-photon RIM, and imaging of thick and scattering media over large fields of view. Finally, we make the first demonstration of event-based DSI. Using event-based detection and random speckle illumination, we obtain optical sectioning from the acquisition, without requiring any post-processing. These technical and technological advances offer new tools and open new perspectives for high speed widefield optical sectioning microscopy applied to biology and the biomedical field.
Keywords : Random illumination,Widefield microscopy, Optical sectioning, Fluorescence microscopy, Super-resolution
