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Biophys J, November 1999, p. 2856-2863, Vol. 77, No. 5
and
Departments of *Physics, #Molecular Biology,
§Chemical Engineering, and ¶Electrical
Engineering, and Princeton Materials Institute,
Princeton University, Princeton, New Jersey 08544 USA
Optical tweezers (infrared laser-based optical traps)
have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser.
Relatively little is known about the origin of this phenomenon. Here we
employed a wavelength-tunable optical trap in which the microscope
objective transmission was fully characterized throughout the near
infrared, in conjunction with a sensitive, rotating bacterial cell
assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at
rates proportional to their transmembrane proton potential (Manson et
al., 1980. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid
and quantitative measure of their metabolic state. Employing this
assay, we characterized photodamage throughout the near-infrared region
favored for optical trapping (790-1064 nm). The action spectrum for
photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and
930 nm. Damage was reduced to background levels under anaerobic
conditions, implicating oxygen in the photodamage pathway. The
intensity dependence for photodamage was linear, supporting a
single-photon process. These findings may help guide the selection of
lasers and experimental protocols best suited for optical trapping work.
Biophys J, November 1999, p. 2856-2863, Vol. 77, No. 5
© 1999 by the Biophysical Society 0006-3495/99/11/2856/08 $2.00
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