Ion transport regulated by protein channels is of great importance in biological systems. Such regulation is highly sensitive and selective. The nature of channel architecture and permeability have been and remain an important area of research. Since the introduction of scanning ion conductance microscopy (SICM), significant effort has been directed toward high resolution topographic imaging of delicate biological samples. Recently, a key feature of SICM, the precise control of probe positions, has been explored with multifunctional probes to obtain local conductance and electrochemical information. In work described here, modifications to SICM were made for the study of ion transport regulated by tight junction proteins in cell monolayers. Modifications include a four-electrode setup (compared to the original two-electrode setup), which allowed localized measurement of heterogeneous conductance on a porous membrane. Further addition of a fifth electrode transformed traditional current recording of SICM to potentiometric measurements, which significantly increased the signal-to-noise ratios and allowed measurement in biological monolayers. With the aid of well-defined samples prepared by focused ion beam, mechanisms of potentiometric-SICM (P-SICM) were studied in detail. P-SICM was then applied to study the interaction of claudin-16 and claudin-19, which are tight junction proteins that control ion transport through cell junctions. With live-cell fluorescence microscopy, cells that expressed unique claudins were identified and targeted with P-SICM. In addition to advances in instrument configuration, the scanning probe of SICM was modified with ion channels inserted in a lipid bilayer at the pipet tip. Such ion channel probes (ICPs) combine the precise position control of SICM with the powerful sensing of ion channels. Approach curves and preliminary imaging performed with ICPs are described in detail. Developments for SICM reported here extend applications available for the study of biological transport.
|Advisor:||Baker, Lane A.|
|Commitee:||Dragnea, Bogdan, Jacobson, Stephen C., Tait, Steven L.|
|School Location:||United States -- Indiana|
|Source:||DAI-B 76/04(E), Dissertation Abstracts International|
|Keywords:||Epithelial cell, Ion channel, Ion transport, Scanning ion conductance microscopy, Tight junction|
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