Single-molecule binding experiments on long time scales

Mark P. Elenko, Jack W. Szostak, Antoine M. van Oijen
2010 Review of Scientific Instruments  
We describe an approach for performing single-molecule binding experiments on time scales from hours to days, allowing for the observation of slower kinetics than have been previously investigated by single-molecule techniques. Total internal reflection fluorescence microscopy is used to image the binding of labeled ligand to molecules specifically coupled to the surface of an optically transparent flow cell. Long-duration experiments are enabled by ensuring sufficient positional, chemical,
more » ... mal, and imagestability. Principal components of this experimental stability include illumination timing, solution replacement, and chemical treatment of solution to reduce photodamage and photobleaching; and autofocusing to correct for spatial drift. Articles you may be interested in Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy Rev. Sci. Instrum. 83, 093708 (2012); 10.1063/1.4752190 Origins of concentration dependence of waiting times for single-molecule fluorescence binding A sensitive and versatile laser scanning confocal optical microscope for single-molecule fluorescence at 77 K Rev. Sci. Instrum. 81, 113705 (2010); 10.1063/1.3499260 Diffusion of carbon nanotubes with single-molecule fluorescence microscopy We describe an approach for performing single-molecule binding experiments on time scales from hours to days, allowing for the observation of slower kinetics than have been previously investigated by single-molecule techniques. Total internal reflection fluorescence microscopy is used to image the binding of labeled ligand to molecules specifically coupled to the surface of an optically transparent flow cell. Long-duration experiments are enabled by ensuring sufficient positional, chemical, thermal, and image stability. Principal components of this experimental stability include illumination timing, solution replacement, and chemical treatment of solution to reduce photodamage and photobleaching; and autofocusing to correct for spatial drift.
doi:10.1063/1.3473936 pmid:20815611 fatcat:obf3oo5wmrh7dgq5a32hojpmw4