Wide-field multiphoton imaging through scattering media

Abstract

Optical imaging has seen exceptional advances over the last two decades. Super-resolution and fast volumetric imaging are now key tools for biological and medical sciences. Whilst these advances have been startling, a remaining challenge in all optical microscopy is to penetrate deeper into tissue. Several approaches have been made to tackle this challenge, including aberration correction or characterisation of the complex media. Although they are remarkable, they are usually very complex, time-consuming and require a "guide star" embedded in the sample. The aim of this thesis is to develop novel microscopy approaches designed to image deeper through biological tissue without correction or characterisation of the scattering medium.

The robustness of temporal focusing to speckle formation upon propagation through scattering media is used to project light patterns onto fluorescent samples located inside or behind a turbid medium. Fluorescent light emitted by the sample is collected in an epifluorescence configuration and the intensity is measured in a single-pixel detection scheme. This technique, termed TRAFIX, achieves an imaging depth of up to 7 scattering mean free path lengths. In addition, as TRAFIX lends itself to compressive sensing it enables high-resolution imaging with minimal photodamage.

Separately, three-photon excitation is implemented for the first time in a light-sheet fluo- rescence microscopy geometry, combining the advantages of both techniques. The addition of propagation invariant beams to this approach, makes it possible to achieve deep penetration in large scattering samples and image at high spatio-temporal resolution over a large field of view.