Magnetic stimulation of neurons and study of membrane structures
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In the work presented in this dissertation, two novel techniques have been developed: i) a magnetic remote stimulation method for remotely triggering ion channel opening and neuron firing, and ii) binned-imaging fluorescence correlation spectroscopy (bimFCS) for probing membrane ultrastructure and studying dynamic changes in membrane structure during signaling events. The development of a magnetic stimulation method is motivated by the fact that the recently developed optical stimulation methods are limited by the inability of light to penetrate deep into biological tissue. In order to achieve magnetic stimulation, 6-nm superparamagnetic MnFe 2 O 4 nanoparticles were synthesized and functionalized to target the plasma membrane of specific subgroup of cells, which express a temperature-sensitive cation channel, TRPV1. When an external alternating magnetic field is applied, the nanoparticles convert energy from magnetic field into localized heat around the cell membrane, opening the TRPV1 to allow calcium influx, therefore triggering membrane potential inversion and action potential firing in neurons. This method is expected to help gain insight into functional connectivity of neuronal networks and provide a mechanism for remote activation of excitable cells in general. bimFCS is a novel technique that combines total internal fluorescence microscopy (TIRF) and fluorescence correlation spectroscopy (FCS), using electron multiplying charge coupled device (EMCCD) for detecting fluorescence intensity fluctuations in each pixel due to the diffusion of membrane protein or lipid of interest. Analyzing the data with different pixel binning sizes allows evaluation of diffusion at various length scales, which yields information about the type of diffusion the molecules undergo as well as the underlying membrane structures with which the molecules interact. Because the diffusion data of different length scales are obtained at the same time in the same area of the same cell, bimFCS permits continuous monitoring of dynamic changes of membrane protein—membrane domain interactions, a unique advantage over other existing techniques. bimFCS is then applied to study the changes of cholesterol-stabilized membrane nanodomains during induced multimerization of both protein and lipid constituents of such domains, as well as the change of ion channel mobility during activation.