Measuring Diffusion in Nanoscale Environments with Single‐Molecule Confocal Fluorescence Microscopy

Single‐molecule detection and diffusion analysis via confocal fluorescence microscopy is a relatively simple and inexpensive way to introduce students to current topics in nanoscience and nanotechnology. We have designed and constructed a confocal microscope that is useful for both laboratory exercises and research investigations. In this article, we discuss three implementations of the microscope within the Chemistry Department. The first involves elementary open‐volume single‐molecule event recordings. The second application addresses molecular surface dynamics of biomaterials and tissue engineering substrates. The third use entails characterizing polyelectrolyte multilayers (PMLs), a new class of thin films, using correlated confocal imaging and single‐molecule diffusion measurements. In each case, fluorescence correlation spectroscopy (FCS) is used to extract diffusion information. For heterogeneous diffusion, specific weaknesses in FCS analysis are identified. Lastly, the overall utility of the instrumentation within the context of undergraduate research and teaching is illustrated.

Keywords

• Polyelectrolyte multilayers,
• Fluorescence correlation spectroscopy,
• Poly(diallyldimethylammonium chloride),
• Polystyrene,
• Single-molecule analysis