Date of Award
digital holographic microscopes, microbial population dynamics, light sheet fluorescence microscope, Arabidopsis thaliana root growth
Quantitative optical measurements at the micron scale have been crucial to the study of multiple biological processes, including bacterial chemotaxis, eukaryotic gene expression and y development. Extending measurements to long time scales allows complete observation of processes that are otherwise studied piecemeal, such as development and evolution. This thesis describes the development of two types of microscope for making long term, quantitative measurements, and the tools for image analysis. The rst device is a digital holographic microscope for measuring microbial population dynamics. It allows three dimensional localization of hundreds of cells within a mm3 sized volume, at micron resolution and an acquisition period of minutes. The technique is simple and inexpensive, which enabled us to construct ten replicate devices for parallel measurements. Each device incorporates precise and programmable control of light and temperature for the microbial ecosystem. Experiments were performed with the green algae Chlamydomonas reinhardtii and the ciliate Tetrahymena reinhardtii, both together and in isolation, and continued for as long as 90 days. The population dynamics exhibited a striking degree of repeatability, despite the presence of added noise in the illumination, spatial gradients of cell density, convection currents and phenotypic changes of both species. The second device is a thin light sheet fluorescence microscope for tracking nuclei in growing roots of the flowering plant Arabidopsis thaliana. The device incorporates a chamber designed to maintain optical quality while providing conditions for root growth. Optical feedback to a translation stage is used to maintain the root tip in the fi eld of view as the root grows by centimeters over several days. Data from a three day experiment is presented to demonstrate the technique. Over 1,000 nuclei were tracked simultaneously, and hundreds of cell divisions were automatically identif ed. The device was also used to image the regeneration of a root tip after surgical excision. The data corroborate earlier investigations at a more detailed level than was previously possible.
Frentz, Zak, "Long-Term Quantitative Microscopy: From Microbial Population Dynamics to Growth of Plant Roots" (2011). Student Theses and Dissertations. 93.