Combined electrical and fluorescence recordings of single molecules in bilayers formed on MECA-opto chips.

Abstract

Molecular processes within lipid bilayers, such as ion channel insertion and gating, can be followed using electrophysiology techniques. With fluorescently labeled molecules, additional features such as protein aggregation and spatial distribution can also be followed. However, correlated electrical and optical measurements, especially at the single-molecule level, have been historically challenging. Factors such as bilayer stability, the timing of protein aggregation and insertion, and the photostability of fluorescent dyes, all work against successful collection of simultaneous electrical and fluorescence records. Here, we describe approaches for assessing lateral diffusion, single-molecule aggregation, and ion channel insertion in bilayers formed on commercially available micro-electrode cavity arrays (MECA-opto chips). These array chips have been specifically designed to enhance the collection of simultaneous electrical and optical records. We compare lipid and ion-channel diffusion results from both fluorescence correlation spectroscopy and wide-field single-molecule tracking experiments. We also use both approaches to assess protein aggregation, spatial distribution, and correlation with insertion currents in the bilayer.