Characterization of Photodamage to Escherichia coli in Optical Traps

Abstract

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.

Implementing both short- and long-working-distance optical trappings into a commercial microscope

“…This value is somewhat low compared to values given in Ref. 23, but the latter were measured for objectives without phase rings. The power losses due to the sample chamber and solution were not estimated and the data given in this work do not account for them.…”

3D visualization of convection patterns in lab-on-chip with open microfluidic outlet

“…It is well known that all common technologies have a strong impact both on the particles and on the surrounding medium, hence limiting the applicability of these techniques. As an example, optical (Neuman et al 1999;Leitz et al 2002;Rasmussen et al 2008) and electromagnetic (Seger-Sauli et al 2005) trapping increase the local temperature through light absorption or the Joule effect, making the trapping environment uncomfortable for cells. This is particularly problematic when the biological material is required to maintain its natural function, and hence needs to be in a condition as similar as possible to its biological environment (Yang et al 2008).…”

Biophysical Tools for Cellular and Subcellular Mechanical Actuation of Cell Signaling

“…While optical tweezers can trap and manipulate individual gold nanorods as slender as 8 nm in diameter (29), heating and the creation of free radicals can have detrimental effects upon cell physiology (30,31). Endogenous particles such as lipid granules can be used as intracellular force handles (32), but the specific biological context of lipid granules and the aforementioned limitations with metallic nanostructures make it challenging to adopt optical tweezers for intracellular manipulation.…”