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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.

Microscopy has been an underpinning technology of the scientific community since it's conception at the end of the 16th century. Significant inventions, which are still in use today, in the form of the fluorescence microscope and the development of fluorescent dyes, have confirmed and enhanced microscopy's place as a staple in science today. However, these advancements would be dramatically reduced if the detector technology had not progressed in parallel. With the development of Single Photon Avalanche Diodes (SPAD) array detectors it is now possible to perform time resolved microscopy on-chip, without the need for any additional counting electronics. This allows for the possibility to perform widefield fluorescent lifetime imaging microscopy (FLIM) due to their fast frame rates and single photon sensitivity. The research presented here is based on the SPAD array technology, SPCImager. The aim is to push the SPCImager platform to its limits by performing widefield single molecule fluorescent lifetime imaging microscopy. The SPCImager is able to perform this due to a dual time-gated imaging modality that allows crude time tagging capabilities over the full array of 320 by 240 pixels. To provide SPCImager with the most optimal and stable conditions, an objective-based Total Internal Reflection Fluorescence (TIRF) microscope set up was designed and developed to allow operation in either CW or Pulsed laser modalities with data being captured on SPCImager and a commercial sCMOS/emCCD simultaneously. To gain a more complete understanding on the deployment of SPCImager's dual time gates, a complete computational model was developed to simulate SPCImager's response to a fluorescent event. By conducting a study into optimising imaging parameters, for a range of simulated events from 1µm carboxylate microspheres to quantum dots and single molecules, it was possible to determine optimal settings required to return accurate and reliable lifetimes. SPCImager was able to perform Widefield FLIM on a range of microspheres before demonstrating its potential and, identifying limitations, when applying this to single molecule widefield FLIM.

Multifocal/multiplane microscopy (MUM) is a technique to acquire simultaneously several planes at sample and obtain axially extended 4D imaging. This is an important characteristic that allows to track fast single molecules/particles three-dimensionally, in real time and over wide axial ranges (≈ 8 μm). MUM avoids possible ambiguous localisations due to the scanning of the imaged plane to acquire a 3D volume over time. For this thesis, a diffraction multiplane system has been characterised to evaluate the impact of different levels of spherical aberration and signal and applied to measure velocity and shear stress fields due to the flow of a liquid around a cell. The spherical aberration has been quantified via the curves of sharpness that can measure the amount of aberrations in images. This has shown that the measured plane spacing grows as the spherical aberration increases. The influence of spherical aberration on image sharpness as a function of emitter axial position could potentially be used to generate correction factors and improve the accuracy on the recovered positions. In terms of performance, the axial range over which the expected axial positions can be calculated with accuracies of at least 100 nm has been shown to vary linearly with the signal level in the studied range. The signal to noise ratio (SNR) threshold below which the axial range goes to 0 μm has been calculated to be 1.23 ± 0.71. It has also been demonstrated that the axial range can be potentially raised by enlarging the plane spacing. Regarding the precision on the axial positions, this varies exponentially with the signal with a decay constant of 0.51 ± 0.10 per SNR unit. This work has generated two equations to predict the expected axial range and precision, given the system parameters are known. Concerning its applications, MUM has been tested to perform micro-particle image velocimetry (μPIV), a technique able to reconstruct the velocity and shear stress fields imposed by a liquid flowing around a cell. The system has been, first, tested in absence of cells, achieving, within 10 μm from the coverslip glass, an accuracy on the calculated velocity of (0.42 ± 0.32) μm/s. This value is slightly worse than that obtained by using a confocal microscope, which is (0.30 ± 0.13) μm/s. Above 10 μm, instead, MUM performance is considerably inferior than that reached with the confocal microscope. In presence of cells MUM has been used for the first time to capture the perturbations to the expected laminar flow, allowing to measure velocities of 30 μm/s and shear stresses of 3 Pa around the observed cell. The reconstructed fields show characteristics similar to those reported in the literature. However, the observation of unexpected velocity and shear stress values indicate a reduction in accuracy caused by false axial localisations.

Cell survival requires the turnover of toxic cellular material and recycling of biomolecules in low nutrient conditions. An efficient degradation system is therefore essential for disease prevention and its dysfunction has been linked to both neurodegeneration and oncogenesis. Bulk degradation is accomplished through the collection of cytoplasmic material in a unique sequestration vesicle, which forms de novo and subsequently deposits cargo in the lysosome for degradation. This process, known as autophagy, therefore requires membrane fusion between the autophagosomal vesicle and the lysosome. SNARE proteins mediate membrane fusion events and therefore their careful regulation ensures the proper organisation of the membrane trafficking network. The SNARE proteins governing autophagosome clearance have been identified as syntaxin 17, SNAP29 and VAMP8 and SNARE assembly appears to be positively regulated by VPS33A. This well established model of SNARE-mediated autophagosome clearance has not, however, been demonstrated within the spatiotemporal framework of the cell and little is known about how VPS33A modulates SNARE function. The research presented in this thesis therefore aims to determine the applicability of the proposed SNARE model within the cellular environment and to investigate the regulatory mechanisms controlling syntaxin 17 function. To accomplish this, carefully validated fluorescence colocalisation and time-resolved fluorescence lifetime imaging techniques were primarily employed. The limitations of these techniques were also considered for data interpretation and a novel prototype SPAD array technology, designed for high-speed time-correlated single photon counting, was trialled for widefield FLIM-FRET. FLIM-FRET revealed that VAMP8 has been incorrectly assigned as the dominant autophagosomal R-SNARE and VPS33A studies evidence a multi-modal regulation of Stx17 that diverges from other studied syntaxin family modulation mechanisms. A new model of SNAREmediated autophagosome clearance is therefore proposed, where syntaxin 17 engages with SNAP29 and VAMP7 to drive membrane fusion with the endolysosome in a manner governed by VPS33A and dependent on the phosphorylation status of syntaxin 17.

Quantum dots (QD) emitting near the telecommunication O-band and excitons believed to be localized on defects in WSe2 monolayers are investigated using optical spectroscopy and resonance fluorescence (RF). The development of light sources emitting around 1300 nm is motivated primarily by the possibility of their use in information communication applications. The results presented in this thesis pave the way towards coherently generated indistinguishable single photons and entangled photon pairs at telecom wavelengths. WSe2 monolayers are highly stable and are characterized by a higher extraction efficiency of photons compared to photon sources embedded in bulk materials. The experiments on confined excitons described here set the stage for the characterization of the effect of valley pseudospin on localized exciton emission properties. A perturbative Coulomb blockade model is applied to telecom wavelength QDs to extract confinement and interaction energies, demonstrating that carriers are in the strong confinement regime. To examine the effect of strong confinement on carrier properties, photoluminescence (PL) spectroscopy of single QDs in the presence of external electric and magnetic fields is performed, and the permanent dipole moment, polarizability, diamagnetic coefficient, and g-factor of excitons localized within them are measured. Temporal measurements on neutral and charged exci- tons are performed, and a bi-exponential decay is observed in the former case, which necessitates a spin-flip interaction with the Fermi sea. RF of telecom wavelength QDs is demonstrated, and numerical simulations are used to characterize the effect of spectral fluctuations resultant from charge noise on RF linewidth. Performing high-resolution spectroscopy, the Mollow triplet is observed and dephasing in the system is shown to be negligible. Second-order correlation function measurements of emission from a localized exciton in a WSe2 monolayer cooled to 4 K under non-resonant and resonant excitation demonstrate its single photon nature. High-resolution PL excitation spectroscopy is used to identify a weakly-fluorescent exciton state blue-shifted from the ground- state exciton. Resonance excitation of the blue-shifted exciton is shown to produce single photons of high purity from the lowest energy exciton state.