Single molecule detection in solution (SMD) based on confocal fluorescence spectroscopy allows investigating the dynamics of proteins under close-to-physiological conditions. It suffers, however, from two limitations: i) The concentration of the molecules under investigation has to be low enough to ensure that only one molecule at a time is located in the focal volume. ii) The observation time is limited to the mean diffusion time of the freely diffusing molecule in the focal volume. We overcome these problems by confining the probe molecules to nanopores in porous alumina oriented parallel to the long axes of the focal volume (Fig. 1).
Figure 1: Schematic diagram of a porous alumina membrane mounted on a Scanning Confocal Optical Microscope. The microscope focus can be adjusted to an area within the membrane. The membrane can be moved along the z-direction (optical axis and long axis of the membrane pores) using a linear actuator.