Bridging Single-Particle Characterisation Gaps of Optical Microscopy in the Nano-Submicron Regime

Doctoral thesis, 2023

As the practical importance of particles in the nano-submicron size regime continues to increase in both biomedical applications and industrial processes, so does the need for accurate and versatile characterisation methods. Optical scattering microscopy methods are commonly used for single-particle characterisation as they provide quick measurements at physiologically relevant conditions with detection limits reaching down to individual biomolecules. However, quantitative particle characterisation using optical microscopy often rely on assumptions about the surrounding media and the
particle, including solution viscosity, boundary conditions, as well as particle shape and material. Since these assumptions are difficult to evaluate, particle characterisation beyond hydrodynamic radius and/or mass remains challenging.

The aim of this thesis is to contribute to bridging the gaps that limit quantitative optical microscopy-based characterisation of individual particles in the nano-submicron regime by both developing new and improving existing microscopy methods. Specifically, in Paper I a method was developed to evaluate the relation between diffusivity and particle size to enable measurements of the hydrodynamic boundary condition. Papers II-V are based around the development of holographic nanoparticle tracking (H-NTA) and extensions thereof, with the intent of using the complex-valued optical field for material sensitive particle characterisation with minimal dependence on the surrounding media. In Paper II, H-NTA by itself was used to characterise suspensions containing nanobubbles and molecular aggregates. In Paper III, the combination of H-NTA with deep learning was used to achieve simultaneous quantification of size and refractive index directly from single microscopy images, which allowed detection of reversible fluctuations in nanoparticle aggregates. In Paper IV, H-NTA augmented with a low frequency attenuation filter, coined twilight holography, was used to investigate the interaction between herpes viruses and functionalised gold nanoparticles in terms of size, bound gold mass, and virus refractive index. In Paper V, the combination of twilight holography and interferometric scattering microscopy (iSCAT) was used to quantify both size and polarizability of individual nanoparticles without the need of detailed knowledge about the surrounding media. Taken together, the presented results in this thesis provide both new insights into heterogenous nanoparticle systems and contributes to narrowing the gap for detailed optical particle characterisation.