Combined Optical Micro Manipulation, Force Microscopy, and Quantitative Phase Imaging for Studying Cellular Membrane Protrusion Dynamics
- Author(s): Sarshar, Mohammad
- Advisor(s): Anvari, Bahman
- et al.
Optical tweezers have become an important instrument in force measurements associated with various physical, biological, and biophysical phenomena. Quantitative use of optical tweezers relies on accurate calibration of the stiffness of the optical trap. In the first chapter, a comparative study of methods in calibrating the stiffness of a single beam gradient force optical trap at trapping laser powers is provided. The Equipartition theorem and Boltzmann statistic methods demonstrate a linear stiffness with trapping laser powers up to 355 mW, when used in conjunction with video position sensing means. The Power Spectral Density (PSD) of a trapped particle's Brownian motion, or measurements of the particle displacement against known viscous drag forces, can be reliably used for stiffness calibration of an optical trap over a greater range of trapping laser powers. Viscous drag stiffness calibration method produces results relevant to applications where trapped particle undergoes large displacements, and at a given position sensing resolution, can be used for stiffness calibration at higher trapping laser powers than the PSD method. The efficacy of optical tweezers is limited in applications where concurrent metrology of the nano-sized structure under interrogation is essential to the quantitative analysis of its mechanical properties and various mechanotransduction events. In the second chapter I report on developing a platform for combined optical micromanipulation and interferometric topography (COMMIT). The all-optical platform delivers pN force resolution in parallel to ≈ 30 nm (or better) axial resolution in biological samples. Plasma membrane tethers are involved in various cellular functions such as motility, cell communications, and transmission of pathogens. In the third chapter, I report on using the COMMIT platform for simultaneous extrusion, force microscopy, and super resolution imaging of membrane tethers from cytoskeleton intact and disrupted HEK239 cells. COMMIT enabled label-free observation of the force-active sub-resolution heterogeneities in tether diameter along its length. I report on observation of cell’s active maintenance of low membrane curvature at the base of the tether to facilitate tether tension relaxation by the Marangoni flow. I also show that the cell is capable of inducing functional shape changes in the tether even in the absence of a functional cytoskeleton.