Ion flow and membrane tension studies with optical tweezers and nanopipettes
This thesis presents a discussion of a novel combination of state-of-the-art experimental techniques into a single microscope setup and the studies this has enabled. Optical tweezers, nanopipettes, and fluorescence were all combined to study the effect of salt species on nanoscale voltage driven flows, the mechanical behaviour of cell membranes, and the electrical activity from various cells. Voltage driven, or electroosmotic, flow was quantified by measuring the piconewton forces of the flow field outside nanopipettes in diff erent salt conditions using optical tweezers. Changing salt conditions revealed new flow behaviour, with flow reversing in CsCl compared to other salts. The role of substrate sti ness in cell membrane tension was investigated using optical tweezers. Optical tweezers were used to trap colloidal particles and then adhere them to the membranes of xenopus laevis retinal ganglion cell axons and NIH 3T3 fibroblasts to pull lipid tethers. Pulling these tethers allowed the e ffective cell membrane tension of these samples to be investigated. These tethers were pulled from cells plated on glass and hydrogels with di fferent elastic moduli. The force response was the same across hydrogels but di fferent on glass, for both neurons and fibroblasts. Fluorescence microscopy was used in-situ to confirm the presence of lipid tethers. Work towards a nanopipette electrophysiology platform was performed, with successful recordings of spontaneous activity taken from rat and mice neuron and astrocyte cultures for longer than 1 hour achieved. Recordings were attempted in brain tissue slices and also performed simultaneously with micro-electrode array recordings for comparison with network activity. Electrophysiological recordings from unmodified escherichia coli bacteria are also demonstrated. The outcome of this work is the advancement of a combined approach using tools from nanoscience to advance the toolset of biophysics. Biophysics concerns itself with complex phenomena, and our findings and demonstrations of method advancement have implications and applications in the understanding of nanoscale transport, cell mechanics, and electrophysiology.