Combining Fluorescence Microscopy on Freestanding Lipid Bilayers with Electrical Measurements

Abstract

The structure, precise operation mechanism and dynamics of ion channels have not yet been elucidated because linking the structure and structure changes to function has remained challenging. We are developing new methods that allow the structure of membrane proteins to be studied in a controlled, native-like environment by simultaneously probing their structure and function. We report a second-generation membrane interferometer (Ganesan et al., Proc. Natl. Acad. Sci. 106, 2008) in which electrophysiology measurements can be performed simultaneously with high-resolution fluorescence microscopy imaging. In the membrane interferometer, a freestanding bilayer is formed over a micropore that is positioned approximately 400 nanometer above a reflective mirror. The mirror allows the use of Fluorescence Interference Contrast microscopy (FLIC) and Variable Incidence Angle-FLIC (VIA-FLIC), two surface characterization techniques that precisely locate the height of fluorescent objects relative to the silicon surface with nm resolution. The second-generation design provides electrical access on both sides and is meant to create a more planar bilayer than the earlier design. Electrophysiology measurements revealed formation of freestanding bilayers with giga-Ohm seals over the micropores that are stable for over 24 hrs. As an initial test we incorporated alpha-hemolysin channels into the bilayers and measured their activity using electrophysiology, while simultaneously imaging the lipid bilayer by FLIC to analyze its curvature. Progress on the incorporation of other fluorescently labeled ion channels will be described.