GSBS Dissertations and Theses

ORCID ID

0000-0003-3363-8791

Publication Date

2019-04-01

Document Type

Doctoral Dissertation

Academic Program

Interdisciplinary Graduate Program

Department

RNA Therapeutics Institute

First Thesis Advisor

David Grunwald

Keywords

microscopy, nanoscopy, fluorescence, cryogenic, cryo-fluorescence, cryoFM, FM, 3DSPEED, SPEED, standardization, PSF, PSF engineering, achromatic, dichroic, multi-color, super-resolution, imaging, imaging standard, optics, adaptive optics, meta-data, meta, MetaMax, calibration, characterization, back-projection, image reconstruction, pseudo-tomography, model-based reconstruction, chromatic aberrations, cryogenic imaging, 4D-PSF, calibration tool

Abstract

Fluorescence microscopy is an essential tool in biomedical sciences that allows specific molecules to be visualized in the complex and crowded environment of cells. The continuous introduction of new imaging techniques makes microscopes more powerful and versatile, but there is more than meets the eye. In addition to develop- ing new methods, we can work towards getting the most out of existing data and technologies. By harnessing unused potential, this work aims to increase the richness, reliability, and power of fluorescence microscopy data in three key ways: through standardization, evaluation and innovation.

A universal standard makes it easier to assess, compare and analyze imaging data – from the level of a single laboratory to the broader life sciences community. We propose a data-standard for fluorescence microscopy that can increase the confidence in experimental results, facilitate the exchange of data, and maximize compatibility with current and future data analysis techniques.

Cutting-edge imaging technologies often rely on sophisticated hardware and multi-layered algorithms for reconstruction and analysis. Consequently, the trustworthiness of new methods can be difficult to assess. To evaluate the reliability and limitations of complex methods, quantitative analyses – such as the one present here for the 3D SPEED method – are paramount.

The limited resolution of optical microscopes prevents direct observation of macro- molecules like DNA and RNA. We present a multi-color, achromatic, cryogenic fluorescence microscope that has the potential to produce multi-color images with sub-nanometer precision. This innovation would move fluorescence imaging beyond the limitations of optics and into the world of molecular resolution.

DOI

10.13028/zq9e-9v53

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Licensed under a Creative Commons license

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This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

Available for download on Wednesday, April 01, 2020

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