DEVELOPMENT OF A MULTIDIMENSIONAL FLUORESCENCE MICROSCOPE USING MULTIPOINT CONFOCAL SCANNING
| dc.contributor.advisor | Fang, Qiyin | |
| dc.contributor.author | Richards, Morgan | |
| dc.contributor.department | Engineering Physics | en_US |
| dc.date.accessioned | 2025-01-24T18:26:24Z | |
| dc.date.available | 2025-01-24T18:26:24Z | |
| dc.date.issued | 2024 | |
| dc.description | The significance of this work is that it bridges the powerful multispectral capabilities of single-point confocal microscopy and the speed and gentle imaging characteristics of multipoint confocal. This will be a powerful technique for acquiring full spectral datasets while preventing photobleaching and phototoxicity. This will enable multilabel measurements to be conducted using a single highly sensitive detector and carve a path to the integration of next-generation time-resolved sensors for multispectral multipoint confocal FLIM microscopy at 1Hz imaging rates. The technology will also have a broader significance to the world as it will reduce the complexity and cost of high-speed multispectral confocal microscopy. Using only a single sensor for readout reduces the financial impact of adopting the technology, as traditional multispectral multipoint microscopy ties the number of spectral bands acquired to the number of sensors used. | en_US |
| dc.description.abstract | Capturing cellular dynamics is key to understanding cell behavior, but this task is challenging due to the weak fluorescence signal in live cells. This signal scarcity becomes more pronounced when divided across multiple contrast dimensions, pushing the boundaries of detector sensitivity. This complexity of measurement is essential for revealing the intricate mechanisms governing cellular function. By using spatial, spectral, and fluorescence lifetime imaging contrasts, we can more precisely isolate species and interactions, uncovering previously hidden aspects of cellular behavior. In this work, we present the development of multiple prototypes for multi-dimensional multipoint confocal microscopy, designed to optimize the use of these faint signals and advance the study of cellular dynamics. Our prototype systems, unmatched in speed and spectral resolution, utilize a pinhole array for efficient confocal multiplexing and dense time-resolved detectors, such as a gated optical intensifier, to measure multipoint confocal time-resolved fluorescence spectra. We demonstrate an enhanced optical design using a 32x32 pinhole array and a SPAD array to capture 960x960 pixel images at a frame rate of 4 Hz. Additionally, we present a 10x10 point multispectral FLIM system, representing the first highly multiplexed multispectral confocal FLIM microscope. A novel optical design further improves the acquisition rate by reducing the sensor readout rate requirements from a quadratic sampling problem to a linear sampling problem. This new optical system can capture 22 spectral bands simultaneously across the 450 nm to 650 nm spectral range at a 1Hz frame rate with a final image resolution of 960x1920. These advancements mark a significant step towards realizing a high-speed multipoint multispectral confocal FLIM microscope and lay the groundwork for future improvements and research. | en_US |
| dc.description.degree | Doctor of Philosophy (PhD) | en_US |
| dc.description.degreetype | Thesis | en_US |
| dc.description.layabstract | Microscopes are essential tools in biology that allow scientists to visualize microscopic structures and processes within cells. Scientists use glowing molecules called fluorophores to color the different parts of the cell to better understand its function. One function of interest is how proteins interact with each other, as this is one of the core processes of a cell's function in life. To measure these interactions, scientists need to make many measurements over time, but these glowing molecules only work for a short period of time before they fade. Building a microscope that can carefully take these measurements all at once and fast enough to see changes would allow careful measurement and might help explain what is happening within the cell. The different methods of measurement are spatial (3D), spectral (Color), dynamic (time), and a special temporal quantum measurement known as the fluorescence lifetime. Together, these measurements form a multidimensional description of the protein’s behavior. In this thesis, I present the tools developed to address these issues and create a fast, multi-dimensional microscope. | en_US |
| dc.identifier.uri | http://hdl.handle.net/11375/30944 | |
| dc.language.iso | en | en_US |
| dc.subject | Multiplexed | en_US |
| dc.subject | Multispectral | en_US |
| dc.subject | Fluorescence Lifetime | en_US |
| dc.subject | Confocal | en_US |
| dc.title | DEVELOPMENT OF A MULTIDIMENSIONAL FLUORESCENCE MICROSCOPE USING MULTIPOINT CONFOCAL SCANNING | en_US |
| dc.type | Thesis | en_US |