ATOMIC FORCE MICROSCOPY WITH A VERSATILE OPTICS SYSTEM FOR APPLICATIONS IN BIOPHYSICS

Cells possess the ability to sense their environment through mechanical means. These interactions include forces from and the extracellular matrix (ECM), fluid flow, and cell-cell adhesion. Abnormal cell mechanical characteristics and failures in a cell’s ability to detect and respond properly to its mechanical environment are implicated in a host of disease states such as muscular dystrophies, emphysema, asthma, hypertension, and cancer. The cell’s cytoskeleton is the interconnecting network of filaments and tubules that mediates forces involved in maintaining cell shape, facilitating motility and bearing mechanical loads. Therefore, tools capable of applying and measuring the forces involved in how cells behave mechanically while simultaneously imaging the parts of the cell responsible for the interaction, such as their cytoskeleton, are an important step toward understanding how cells function and understanding diseases. In this project, I expand upon previous work combining the atomic force microscope (AFM), with its versatility of operation and range of detectable forces, with the high-quality fluorescence imaging of light sheet microscopy by adding a versatile optics system, called a Versatile Illumination Engine With a Modular Optical Design (VIEW-MOD). VIEW-MOD is an optical engine designed to incorporate multiple advanced fluorescence microscopy techniques into a single optical pathway. It is capable of rapid switching between techniques and is completely computer controlled. It is the first instance of controlled illumination for point, broad and light sheet all realized in one illumination pathway. Here I develop and apply the system’s capability of two-color light sheet volume scanning with simultaneous AFM data to phagocytosis and find podosome-like structures form under the AFM bead tip during phagocytosis. I demonstrate the first AFM and phagocytosis experiment, and inform mechanical models of phagocytosis from the results for FcɣR-mediated and Dectin-1 mediated phagocytosis.Doctor of Philosoph