Simultaneous single photon counting and ion channel recording from free-standing lipid bilayers on a microstructured array.

Abstract

Fluorescence microscopy and, in particular, single molecule optical spectroscopy is of great potential value in characterizing the structural dynamics of membranes and membrane proteins. A particular challenge is to combine such high-resolution optical measurements with high-resolution voltage clamp electrical recordings that can provide direct information on single ion channel gating a block by drugs and analytes. Here, we report on the use of a novel chip-based, 2 × 2 arrangement of microelectrode cavities with borosilicate glass optical windows which facilitates optical access on an inverted microscope with water or oil-immersion objectives of high numerical aperture to horizontal free-standing lipid membranes, while controlling membrane voltage and recording currents using individual micropatterned, ring-shaped Ag/AgCl-electrodes to perform time-resolved single photon counting on free-standing membranes spanning sub-nanoliter cavities. Single channel activity induced by the fluorescently labelled pore-forming peptide ceratotoxin A was simultaneously acquired. This device allows for rapid formation of four membranes that are simultaneously monitored electrically using a four-channel amplifier and can be sequentially optically addressed, greatly reducing the time needed for successful experimentation. During our experiments, we noted autofluorescence of the borosilicate to be a limiting factor for wide-field low-intensity fluorescence recording, but this can likely be circumvented by using quartz glass instead. In summary, the novel device increases the likelihood of realizing the long standing ambition to correlate structural and functional dynamics of single membrane proteins on the single molecule level.