Nonlinear microscopy methods for imaging and particle tracking in thick biological specimen
Access full-text files
Date
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Optical microscopy techniques such as single-particle tracking and high-resolution (<500 nm) imaging are critical tools for the advancement of biological research. However most high-resolution optical techniques utilize a camera-based or confocal-based detection scheme, which limits the working distance into samples to approximately 10 μm due to light scattering. Nonlinear excitation methods, such as two- and three-photon microscopy, have enabled imaging in thick and scattering samples due to their longer excitation wavelengths and absence of spatial filtering. However, nonlinear excitation is rarely utilized for single-particle tracking as it traditionally offers slightly worse resolution than the aforementioned methods. This dissertation presents the progress made towards adapting nonlinear excitation for high-resolution biological study at scales ranging from single-molecules up to entire tissues. We describe a novel single-particle tracking microscope based upon multiplexed nonlinear illumination, coined TSUNAMI. Single-particle tracking with nanometric resolution using TSUNAMI is demonstrated in live cells and spheroid tumor models to unprecedented depths of 200 μm. Several new long wavelength excitation laser sources are detailed which provide superior image penetration depth compared with traditional sources. Furthermore, we detail a newly discovered form of nonlinear excitation, based upon a two-color, three-photon absorption process, and discuss potential benefits of this new excitation regime. The systems and methods developed in this work will provide life scientists with a powerful toolset for the future of biological research.