Characterization of photodamage induced by optical tweezers

Summary form only given. Optical tweezers or optical traps are a unique tool capable of manipulating and controlling biological molecules in vitro and in vivo. A major drawback to using optical traps is the potential for damage produced by the trapping laser in the sample. Optical damage limits the exposure for any trapped specimen and has proved to be a significant problem for optical traps used in vivo. Despite these obvious problems, little work has been carried out to characterize or quantify damage produced in optical traps. This has been due, in part, to the unavailability of a convenient biological system to assess the damage. Here, the authors describe a novel biological assay employed in conjunction with an optical tweezers set up to systematically study damage produced in optical traps, The assay is based on tethering cells of Escherichia coli (strain KF952) to a glass coverslip by a single flagellar filament. Such cells rotate at speeds that are proportional to their transmembrane potential (protonmotive force), thus providing a direct and quantitative measure of their state of metabolic health. Tethered cells are held by the optical trap and periodically released to monitor their rotation frequency. The rotation rate is thereby established as a function of time in the trap. The ability to monitor continuously the metabolic health of the cell allows one to observe how the damage occurs as a function of time and to quantify this optical damage. This procedure is carried out for many wavelengths to establish the wavelength dependence of optical damage. The wavelength dependence shows a damage minimum at 830 nm, where cells live much longer than at 1064 nm, the later wavelength being most commonly used for optical traps.