Xiangyi LI will defend her thesis entitled “Widefield linear and non-linear optical microscopies using random illumination and temporal focusing” on Thursday, January 22nd at 02:00 p.m. in room Pierre Cotton of Institut Fresnel.
Composition of the Jury :
– Marc GUILLON, SPPIN, Reviewer
– Eirini PAPAGIAKOUMOU, Institut De La Vision, Reviewer
– Hilton BARBOSA DE AGUIAR, Kastler Brossel Laboratory, Examiner
– Hervé RIGNEAULT, Institut Fresnel, Examiner
– Martin OHEIM, SPPIN, Jury President
– Anne SENTENAC, Institut Fresnel, Thesis supervisor
Abstract : Wide-field optical microscopy, in which the whole sample is illuminated in one shot and the image is recorded on a camera, is widely used for observing micrometric and submicrometric structures due to its speed, robustness, and minimal impact on samples. However, it suffers from two major limitations: limited transverse resolution (at best, half the wavelength) and, more critically, a lack of optical sectioning. Because the sample is also illuminated in depth, light from out-of-focus planes degrades image contrast, making the technique ineffective for thick samples. This thesis explores how random speckle illuminations and temporal focusing can introduce optical sectioning in wide-field microscopy, particularly for one and two-photon excitation fluorescence microscopy and for nonlinear imaging. First, the work investigates Random Illumination Microscopy (RIM) for one-photon fluorescence. RIM is a widefield microscopy method that uses the variance of mul-tiple images acquired under different speckle patterns to numerically reconstruct a super-resolved, optically sectioned image. While RIM was originally developed for 2D imaging, this thesis extends the algorithm to 3D, introducing an iterative deconvolu-tion procedure tailored for images with few optical planes. The 3D approach yields better resolution and contrast than slice-by-slice 2D reconstruction. Next, the study addresses two-photon excitation fluorescence microscopy (2PM), which typically relies on scanning a tightly focused beam for sub-micrometer res-olution and optical sectioning. However, the scanning modality proves slow when imaging large fields of view. To overcome this issue, we extended RIM principles to two-photon wide-field microscopy (2PE-RIM) . We theoretically and experimentally demonstrate an improved resolution and optical sectioning. A second approach con-sisted in developing a novel excitation scheme for the scanning modality. We showed that a focused speckled beam is able to form a 2P excitation volume with a transverse width and axial width of a few micrometers. This illumination scheme is susceptible to provide faster scans of large fields of view (at the cost of a reduced resolution). In the third chapter, we also explore the potential of Temporal Focusing (TF), for generating optical sectioning in non-linear widefield microscopy. To create a tem-porally focused beam, a pulse is sent onto a grating and the dispersed wavelengths are recombined in such a way that their interference are constructive only at the focal plane. While standard grating-TF requires high magnification, combining it with random speckle illumination (roughness-grating TF) enables micrometer-level optical sectioning at low magnification, suitable for large fields of view. We showed that combining roughness-grating TF with RIM improved both the transverse and axial resolutions of widefield 2PM.
Finally, we studied the interest of TF illumination for Coherent Anti-Stokes Raman Scattering (CARS) microscopy, a label-free technique for probing specific chemical bonds. We showed theoretically and experimentally that by temporally focusing both the pump and Stokes beams, we could obtain optically sectioned CARS images in a widefield configuration.
Keywords : Non-linear microscopy, Super-resolution, Temporal focusing, Two-photon microscopy, Optical sectioning, Coherent anti-Stokes Raman scattering microscopy
