With the wide-spread adoption of exogenous fluorescent indicators – and more recently genetically encoded fluorescent proteins – over the past two decades, there exists a diverse chemical toolkit with which to probe biological systems. Individual cell types and sub-cellular compartments can be targeted in an increasingly wide range of model organisms. However, imaging these samples is often an exercise in balancing the needs of any given experiment against the constraints of the chosen imaging technology. For example, a volume of brain tissue is host to neurons, glia, vascular compartments and red blood cells that all occupy discrete locations in 3D space, but must work together to support healthy organ function. Single-cell activity on the order of milliseconds can trigger downstream processes that unfold over the course of multiple seconds or even minutes. The development of a technique capable of providing depth-resolved, volumetric imaging with scalable spatiotemporal resolution is crucial to developing a proper understanding of such biological systems.
Bottlenecks in the throughput of existing technologies stem from a combination of inefficient illumination and volume acquisition strategies, and insufficient sensor read-out speeds. Light sheet microscopy is a promising solution, but individual designs tend to be highly specialized to specific types of samples and do not easily adapt to a wide range of experimental settings. In this thesis, I detail my work in developing swept, confocally-aligned planar excitation (SCAPE) microscopy from a first-generation prototype into a versatile, easy-to-reproduce, easy-to-use system for high-speed, 3D imaging.
The first chapter introduces the challenges of designing optical systems capable of high-speed, volumetric imaging. An introduction to design choices faced in the construction of fluorescence microscopes, and current approaches to 3D imaging are discussed. The second chapter describes the progression from the 1st to 2nd generation SCAPE system. Improvements made through ray-tracing models and an enhanced optomechanical design are described, and results from this system in a number of model organisms are presented. The third chapter presents results from a range of biological applications to which SCAPE microscopy has been applied. Work in imaging the zebrafish heart to demonstrate the system’s improved imaging speed, the C. elegans to show the system’s resolution, and finally a number of examples of large field-of-view and high-resolution structural imaging are all described. Finally, the fourth chapter concludes with an overview of the work that lies ahead to both further develop of SCAPE microscopy, as well as to bring the existing system’s strengths to bear in a wider range of environments.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/d8-bf45-yj52 |
| Date | January 2019 |
| Creators | Voleti, Venkatakaushik |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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