Development of an experimental apparatus for simultaneous observation of optical and electrical signals from single 1on channels

We have developed an experimental apparatus for the simultaneous measurement of optical and electrical properties of single ion channels. The microscope designed for single-molecule detection was combined with the artificial bilayer single-channel recording system. The artificial membranes were formed horizontally in an aqueous environment or on a thin agarose layer. Single molecules in the bilayer were observed under an epi-fluorescence or an objective-type total internal reflection fluorescence micoscope. Minute currents across the bilayers were measured by a patch clamp amplifier under voltage clamped conditions. The apparatus developed in this study was sensitive enough to detect the optical signals from single-fluorophores in the bilayer simultaneously with the single-channel current recording. Using this apparatus, the following results were obtained: (i) Brownian motion of single-lipid molecules in the bilayer was observed. (ii) Cy3-alamethicin molecules were incorporated into the bilayer and the fluorescence image was recorded simultaneously with the channel current recording at the single-channel level. (iii) The liposome fusion process to the bilayer could be observed at the single molecule level. (iv) Various types of channels could be incorporated into the bilayers on agarose. The properties of the channels were identical to those determined in the bilayer formed in an aqueous environment.