Adaptive optics for volumetric microscopy

Abstract

Volumetric microscopy allows three dimensional information to be captured in a single image, but the imaging quality can be adversely affected by optical aberrations. We investigate the robustness and efficiency of optical aberration compensation for widefield and volumetric microscopy using iterative image-based - or sensorless - modal adaptive optics (AO). In this thesis, we use extensive numerical modelling to show that the limiting factors for an accurate modal correction are measurement linearity and non-linear modal crosstalk. We demonstrate that estimating 4 metric values per mode provides overall better and more robust estimates of modal amplitudes, regardless of initial Strehl or signal-to-noise ratio, as well as minimising the total number of photons used for correction. We show that accurate aberration estimation can be obtained for up to 1 radians RMS of initial aberration; and an excellent AO correction can still be obtained beyond this range when an appropriate optimisation algorithm is employed. In addition we quantify the impact of out of focus light and three dimensional sample structure on the correction capability. We also present an adaptive optics correction system employed on an Olympus microscope, and demonstrate image sharpening on both two dimensional and three dimensional images.