Advancing the limits of mass photometry

Thesis

In recent years, technological developments have enabled the detection and imaging capabilities of light scattering microscopy to reach single molecule sensitivity. Here, we first present many of the recent technological developments in the broader field of light scattering microscopy. Amongst the myriad of techniques reviewed, mass photometry presents a novel method to image, locate, and identify single biomolecules in solution by way of optically measuring their mass. These attributes have allowed mass photometry to become a widely-used bioanalytical technique across a variety of biomolecular applications. However, the key performance parameters, namely the detection sensitivity and the mass resolution, are limited by a number of experimental factors. The manifestation of these limitations presents a wide berth between the currently achievable detection parameters of mass photometry and the theoretically attainable.

This thesis presents a thorough investigation into the experimental limitations of mass photometry, with the goal of understanding and ultimately overcoming them by means of novel experimental methods. A combination of noise-reduction procedures and contrast enhancement by way of phase-offset optimisation led to a significant improvement in the achieved mass resolution. However, it was found that at the limits of mass photometry, a particle-like, dynamic speckle noise is ever-present and defines the lower bound of the achievable detection sensitivity, as well as reducing the mass resolution. This noise was found to be located near to the sample region and its behaviour was probed by means of original experimental methods.

We conclude with the introduction of two novel optical approaches to improve the signal-to-noise ratio of mass photometry measurements – anisotropic image magnification and image replication. The work presented here proves the concept of both of these techniques as viable approaches to improve the performance parameters of mass photometry and provides a foundation for further work to build upon.

Authors: Priest, L

• DOI: 10.5287/ora-kqyvxq7zy
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: mass photometry; interformetric scattering microscopy; iSCAT
• Subjects: Biophysics; Rayleigh scattering; Microscopy