Wavefront shaping for stimulated emission depletion microscopy

Thesis

Super-resolution microscopy has significantly expanded the scope of application of biological microscopes by allowing the resolution of features smaller than the diffraction limit. Several super-resolution microscopy techniques exist, among which stimulated emission depletion (STED) microscopy has become a tool of choice for many biological applications, particularly in living specimens. STED has also proven useful as a tool for biophysics, thanks to its capability to be combined with fluorescence correlation spectroscopy (FCS). Unfortunately, the excellent resolution of STED microscopes comes at the cost of an increased experimental complexity, which limits the scope of potential applications. This is particularly true in deep, three-dimensional specimens, where optical aberrations and undepleted background significantly reduce measurement quality. In this thesis, we developed methods based on wavefront shaping to overcome these issues, for STED imaging and STED-FCS. We used for this an adaptive STED microscope equipped with a spatial light modulator for wavefront shaping, for which we first developed a bespoke calibration protocol. We particularly optimised the performance of z-STED, a STED confinement mode that increases the axial resolution and was so far scarcely used due to its sensitivity to optical aberrations. Using this newly calibrated z-STED microscope, we investigated the dynamics and structure of the plasma membrane of living cells. To image deeper in aberrating specimens, we developed a novel aberration correction method, based on a novel image quality metric developed in our lab. This method was then adapted to establish a new aberration correction method for STED-FCS, which we could use to increase the performance of z-STED-FCS in solution as well as in cells. Finding suboptimal performance of z-STED-FCS even after aberration correction, we investigated the origins of background noise in STED-FCS in 3D and found that it could be minimised by means of coherent-hybrid STED created with a bivortex phase mask.

Authors: Barbotin, A

• DOI: 10.5287/ora-qaykj99gz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: microscopy; FCS; STED-FCS; STED
• Subjects: Lasers in biophysics; Biophysics; Optics