University of Cambridge
Advances in volumetric super-resolution microscopy and single-particle tracking
Abstract
dc:description.abstractSingle-molecule localisation microscopy (SMLM) has enabled optical microscopy to probe biological structures and processes at the nanometer scale. To accommodate the inherently 3D nature of biological structures, 3D-SMLM techniques---such as the astigmatic and double-helix point spread functions (PSFs)---have been developed. These techniques encode the lateral and axial positions of single fluorophores into the PSF shape but at the cost of reduced spatial and temporal resolution. This limitation restricts the compatibility of 3D-SMLM with established labeling protocols, increases experimental duration and reduces biological throughput. Thus, accelerating 3D-SMLM is essential for broader applicability in the life sciences. Single-molecule light field microscopy (SMLFM) is a novel 3D-SMLM approach that uses a microlens array in the back focal plane of an optical microscope to capture fluorescence from multiple perspectives. Conceptually, SMLFM improves the density limit of SMLM through three key factors: 1. The PSF footprint resembles a simple and compact 2D Gaussian function that can be better distinguished at high density compared to complex engineered spatial patterns. 2. Laterally overlapping fluorophores can be better distinguished as a result of the parallax-based optical model. 3. An optical redundancy in the number of perspective views required for 3D reconstruction increases the range of working densities. This thesis documents the characterisation, validation, and application of the first hexagonal SMLFM platform. In brief, Chapter 3 describes a workflow for establishing SMLFM design parameters, useful for researchers looking to build their own platforms. Furthermore, optical validation and characterisation protocols benchmarked SMLFM in both static and dynamic imaging modes over an 8 μm axial range. Chapter 4 describes the quantitative comparison between SMLFM and other 3D PSFs, demonstrating similar resolving power and imaging speed between SMLFM and astigmatism, and a ten-fold speed improvement over the state-of-the-art double-helix PSF. Various imaging applications of SMLFM are then presented. Chapter 5 explores the stoichiometry of the B-cell receptor on primary mouse B cells and the spatial enrichment of the PD-1 receptor relative to the membrane morphology of T cells. Chapter 6 applies SMLFM in a single-particle tracking modality to quantify the diffusive states of Golgi-localised proteins, RAB6 and ACBD3, and reports on a biophysical investigation into the effect of transmembrane domain length on protein secretion.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Daly, Sam
- Advisor dc:contributor.advisor
-
- Lee, Steven Frank
Subjects
dc:subject × 7Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
- 0000-0002-8559-3161
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/376474