Single-molecule orientation estimation using a polarisation camera

Abstract

This thesis presents the development of a new optical imaging method for measuring the orientation and position of single fluorescent molecules. The method - named POLCAM - is based on the use of a polarisation camera. Current methods for single-molecule orientation estimation require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption of these methods for biological applications. In this work, a polarisation camera is used to dramatically simplify the experimental setup required for single-molecule orientation estimation; the method can be easily implemented on any wide-field fluorescence microscope, making it highly accessible to biological research labs. As polarisation cameras are not designed with highly sensitive applications like single-molecule detection in mind, this work includes an in-depth characterisation of a commercial polarisation camera, and a comparison with the state-of-the-art cameras that are typically used in single-molecule microscopy. This work also includes the development of optical simulation software to generate realistic single-molecule polarisation camera images. This simulation software is then used to validate and test the limits of the data analysis software developed as part of this work, including a fast algorithm based on Stokes parameter estimation. The developed algorithm can operate over 1.000-fold faster than the state of the art, enabling near instant determination of molecular orientation. Finally, to illustrate the potential of POLCAM in the life sciences, we applied our method to study alpha-synuclein fibrils and the actin cytoskeleton of mammalian cells.