Single-molecule binding experiments on long time scales.
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.
Elenko
Mark P
MP
Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, USA.
Szostak
Jack W
JW
van Oijen
Antoine M
AM
D052157:Aptamers, Nucleotide / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D013696:Temperature; D013997:Time Factors
D052157:Aptamers, Nucleotide; D008024:Ligands; D006160:Guanosine Triphosphate