Abstract
Single-molecule super-resolution microscopy is an emerging technique for nanometer-scale fluorescence imaging, but in vitro single-molecule imaging protocols typically require a constant supply of reagents, and such transport is restricted in constrained geometries. In this article, we develop single-molecule micelle-assisted blink (MAB) microcopy to enable subdiffraction-limit imaging of nanochannels with better than 40 nm accuracy. The method, based on micelles and thiol-related photoswitching, is used to measure nanochannels formed in polydimethylsiloxane through tensile cracking. These conduits are reversibly size-adjustable from a few nanometers up to a micrometer and enable filtering of small particles and linearization of DNA. Unfortunately, conventional techniques cannot be used to measure widths, characterize heterogeneities, or discover porosity in situ. We overcome the access barriers by using sodium dodecyl sulfate (SDS), an ionic surfactant, to facilitate delivery of Cy5 dye and β-mercaptoethanol reducing agent in the confined geometry. These SDS micelles and admicelles have the further benefit of slowing diffusion of Cy5 to improve localization accuracy. We use MAB microscopy to measure nanochannel widths, to reveal heterogeneity along channel lengths and between different channels in the same device, and to probe biologically relevant information about the nanoenvironment, such as solvent accessibility.