Single-molecule microscopy using tunable nanoscale confinement

Abstract

We present the design, construction and implementation of a modular microscopy device that transforms a basic inverted fluorescence microscope into a versatile single-molecule imaging system. The device uses Convex Lens- Induced Confinement (CLIC) to improve background rejection and extend diffusion-limited observation time. To facilitate its integration into a wide range of laboratories, this implementation of the CLIC device can use a standard flow-cell, into which the sample is loaded. By mechanically deforming the flow-cell, the device creates a tunable, wedge-shaped imaging chamber which we have modeled using finite element analysis simulations and characterized experimentally using interferometry. A powerful feature of CLIC imaging technology is the ability to examine single molecules under a continuum of applied confinement, from the nanometer to the micrometer scale. We demonstrate, using freely diffusing λ-phage DNA, that when the imposed confinement is on the scale of individual molecules their molecular conformations and diffusivity are altered significantly. To improve the flow-cell stiffness, seal, and re-usability, we have innovated the fabrication of thin PDMS-bonded flow-cells. The presented flow-cell CLIC technology can be combined with surface-lithography to provide an accessible and powerful approach to tune, trap, and image individual molecules under an extended range of imaging conditions. It is well-suited to tackling open problems in biophysics, biotechnology, nanotechnology, materials science, and chemistry.