Waveguide-based Excitation for High-throughput Imaging

Abstract

Chip–based fluorescence imaging is an emerging field where the fluorophores in a sample are excited by the evanescent field generated by waveguides. The aim of this thesis is to explore how waveguide–based excitation can benefit super–resolution optical microscopy, as well as investigate other imaging and spectroscopic applications which can benefit from the same platform.

For all imaging and spectroscopic applications, a background signal can be detrimental to the results. The first part of this thesis is thus an experimental evaluation of the background signal from two common waveguide platforms, silicon nitride (Si3N4) and tantalum pentoxide (Ta2O5), to evaluate their potential use for imaging and sensing applications within the visible range of light. It is shown that both platforms perform approximately equally well at 640 nm excitation, but at shorter wavelengths Si3N4 has an increasing fluorescent background. The impact of the increasing background signal at shorter wavelengths for Si3N4 on direct stochastic optical reconstruction microscopy (dSTORM) is shown to be problematic as it reduces the localisation precision. Chip–based resonance Raman spectroscopy of haemoglobin with 532 nm excitation using a Ta2O platform was performed.

dSTORM is a popular super–resolution optical microscopy technique due to its ease of use and extremely high resolution. To reduce out–of–focus light in order to increase the contrast and resolution, total internal reflection fluorescence excitation is often preferred to epi–illumination. This is commonly generated through specialised lenses which have a small field–of–view. In this thesis, I show how using waveguide–based excitation can enable imaging of unprecedentedly large areas for nanoscopy. The chip–based nanoscope exchanges the excitation pathway and sample holder of a traditional microscope with an integrated photonic chip to enable total internal reflection fluorescence imaging. This thesis exhibits both diffraction–limited and super–resolution imaging of 500 µm × 500 µm image of liver sinusoidal endothelial. Furthermore, a modified system was developed to combine the super–resolution imaging with quantitative phase imaging for three–dimensional morphological imaging. This system enabled nanoscale measurement of both the of sieve plates in liver sinusoidal endothelial cells and the size. In the latter part of the thesis, I show how inhomogeneous excitation from waveguides can be used for computational imaging. In particular, the deterministic nature of the patterns is used to enhance the resolution of images. This shows how a waveguide platform not only can enhance existing imaging techniques, but also offer novel approaches as well.