Date of Award
Open Access Thesis
Robert L. Price
Optical microscopy resolution is limited by the wavelengths of light and the series of microscope lenses and other optical components used to create a magnified image of cell structures in a sample. Often the cell structures are smaller or closer together than the resolution limits of a light microscope. In 2015 the Boyden group at the Massachusetts Institute of Technology (MIT) created a sample preparation technique, expansion microscopy which involves embedding biological samples in a crosslinked, swellable, hydrogel polymer that allows for uniform physical separation of cell components so that they can subsequently be resolved by light microscopy. Using the protein retention variant of expansion microscopy, these experiments evaluated the efficacy of the technique. Forty-micron thick rat hippocampal specimens were labeled GFAP and NeuN antibodies to image the neurons and astrocytes within the tissue. The expansion process produced a 4.7-fold physical expansion of the hippocampal slices. The process was also evaluated using 50 – 100 um thick samples of mouse cardiac tissue, labeled to visualize the cytoskeleton, intercalated discs, and cellular nuclei. However, due to the strong structural integrity of cardiac tissue expansion was inconsistent and incompatible with cardiac tissue samples. Variations to the procedure are required to compensate for the rigidity and anisotropic nature of cardiac tissue. One challenge faced when expanding the samples was creation of a consistent and uniform expansion gel and handling of the fragile embedded tissue. To alleviate these difficulties, we designed a new gelation chamber that allowed for more consistent sample preparation.
Ferri, A.(2020). Expansion Microscopy: A New Approach to Microscopic Evaluation. (Master's thesis). Retrieved from https://scholarcommons.sc.edu/etd/6034