Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item Development of an active/adaptive laser scanning microscope(Montana State University - Bozeman, College of Engineering, 2018) Archer-Zhang, Christian Chunzi; Chairperson, Graduate Committee: David L. DickensheetsLaser scanning techniques such as confocal microscopy and two-photon excitation fluorescence microscopy (TPM) are powerful tools for imaging biological samples with high resolution, offering three-dimensional (3D) visualization of the behavior of cells in their natural environment. Traditionally, the 3D images are acquired from 2D image stacks with focusing depth controlled through mechanical movement of the specimen relative to the objective lens. The slow mechanical movement (~<20Hz) does not allow the spot of light to be scanned axially sufficiently fast to monitor cell:cell and cell:environment interactions in real time over hundreds of microns in all three dimensions. A fast focus control mirror supports agile scan patterns such as vertical or oblique planes or even arbitrary surfaces, minimizing the time and photo damage required to monitor features of interest within the 3D volume. Because aberrations cause image quality to decrease as the focal point of the beam penetrates deeper into the sample, adaptive optics can enhance resolution and contrast at depth for confocal microscopy and TPM. Combining a fast focus control mirror with a fast aberration correcting mirror leads to a flexible platform called the active/adaptive laser scanning microscope, capable of aberration-corrected beam scanning throughout a 3D volume of tissue. This opens up the possibility of fully corrected, variable-depth imaging along oblique sections or more complex user-defined surfaces within a single image frame.Item Wide field of view two-photon excited fluorescence imaging, theory and applications(Montana State University - Bozeman, College of Letters & Science, 2016) Stoltzfus, Caleb Ray; Chairperson, Graduate Committee: Aleksander RebaneTwo-photon excited fluorescence (2PEF) is a unique photophysical process that has benefited many diverse areas of science. Imaging the 2PEF signal offers numerous intrinsic benefits, including low background scattering, high sample photo-stability, and high excitation selectivity. The 2PEF signal has a nonlinear dependence on excitation intensity, which has proven to be extremely useful for high resolution, three dimensional microscopy. This same nonlinear dependence, in conjunction with the typically low probability of two-photons being simultaneously absorbed, also makes 2PEF imaging difficult to scale, leaving most two-photon microscopes with a field of view (FOV) limited to less than a few mm 2. This effectively limits the benefits of the unique properties of 2PEF imaging to microscopic applications. This dissertation explores the development and application of a wide FOV 2PEF imaging technique, where a FOV as large as 10 cm 2 is achieved by increasing the peak photon flux of the excitation source, and expanding the illumination region. The use of this imaging technique for the in depth characterization and optimization of fluorescent proteins (FPs), as well as taking high contrast images of fingermarks is described. This new wide FOV 2PEF imaging technique greatly expands the usefulness of the unique photophysical properties of 2PEF and allows for sensitive, high contrast 2PEF imaging on a much larger scale than was previously possible.