Development and characterisation of waveguide total internal reflection fluorescence microscopy

Single-molecule fluorescence microscopy is a growing and powerful tool for studying molecular biophysics. However, the field faces several barriers to entry, limiting its uptake. Open-source microscopy is an emerging field where published setups seek to address the barriers to microscopy entry, including single-molecule fluorescence microscopy. LED-based waveguide total-internal reflection fluorescence (WG-TIRF) excitation is a promising, under-researched method highly suited to an open-source design format.

This thesis aims to research the WG-TIRF excitation method, develop novel characterisation methods for the power density and detection sensitivity of WG-TIRF devices, and apply identified improvements to design a WG-TIRF device. A collaboration was established with 454 Bio to address these aims using prototype versions of their now open-source WG-TIRF microscope.

In addressing the development of a power density characterisation method, a technique using characterised photobleaching rates at known power densities was used to establish an approximation of power density over the whole field of view. This method was extended to function on a grid layout, allowing for analysis of spatial variation in power density and understanding the Lambertian emission effects of LEDs on the excitation profile of different optical configurations.

In addressing the second aim, a device sensitivity characterisation method was developed that used fluorescent microspheres to determine the single-fluorophore detection capabilities of a WG-TIRF system. This method provided a quantitative method to compare the changes in various system parameters, allowing for improving WG-TIRF devices, as seen in changes to the 454 Bio WG-TIRF device.

Identified design factors and improvements from aims one and two were applied in designing a WG-TIRF excitation device, which works with a standard inverted fluorescence microscope. The presented designs allow for the building of the device, and the geometric proofs increase the understanding of the device's optical properties and allowed for a model for power density within the LED WG-TIRF system to be developed. The research of this thesis shows that WG-TIRF is a viable fluorescence microscopy method and an accessible entry point into the field, the development of which will require the characterisation methods developed in the thesis.