Comet Assay Measurements of DNA Damage in Cells by Laser Microbeams and Trapping Beams with Wavelengths Spanning a Range of 308 nm to 1064 nm

Abstract

DNA damage induced in NC37 lymphoblasts by optical tweezers with a continuous-wave Ti:sapphire laser and a continuous-wave Nd:YAG laser (60-240 mW; 10-50 TJ/m2; 30-120 s irradiation) was studied with the comet assay, a single-cell technique used to detect DNA fragmentation in genomes. Over the wavelength range of 750-1064 nm, the amount of damage in DNA peaks at around 760 nm, with the fraction of DNA damage within the range of 750-780 nm being a factor of two larger than the fraction of DNA damage within the range of 800-1064 nm. The variation in DNA damage was not significant over the range of 800-1064 nm. When the logarithm of damage thresholds measured in the present work, as well as values reported previously in the UV range, was plotted as a function of wavelength, a dramatic wavelength dependence became apparent. The damage threshold values can be fitted on two straight lines, one for continuous-wave sources and the other for pulsed sources, irrespective of the type of source used (e.g. classical lamp or laser). The damage threshold around 760 nm falls on the line extrapolated from values for UV-radiation-induced damage, while the data for 800-1064 nm fall on a line that has a different slope. The change in the slope between 320 and 340 nm observed earlier is consistent with a well-known change in DNA-damaging mechanisms. The change observed around 780 nm is therefore suggestive of a further change in the mechanism(s). The data from this work together with our previous measurements provide, to the best of our knowledge, the most comprehensive view available of the DNA damage produced by microfocused light.

After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

“…The peak at 365 nm was filtered with a combination of two colour glass filters UG1 und KG1 (both obtained from Schott, Germany). Thorough definition of the wavelength is mandatory because there is a strong dependence of light induced DNA damages on wavelength (Mohanty et al, 2002;de With and Greulich, 1995). The resulting fluence rate of 535 W/m 2 was measured with a Solarscope UV Radiometer (SolarTech, UK) and it was assured that no irradiation below 340 nm passes through the filters.…”

Optoelectronic Tweezers for the Manipulation of Cells, Microparticles, and Nanoparticles

“…Thus, the trap is only stable in three dimensions if the axial gradient force near the focal point is stronger than the forces from radiation pressure. A typical optical trap requires an intensity of approximately 10 7 mW/cm 2 , which requires high-power lasers, and can damage some types of particles, including cells (Konig et al, 1996, Neuman et al, 1999Mohanty et al, 2002). In addition, the generation of sufficient optical gradients requires high numerical aperture objective lenses to focus the laser; this results in a limited optical manipulation area, reducing parallel manipulation capabilities, especially for larger microscale particles such as cells.…”

Reduction in DNA Synthesis During Two-photon Microscopy of Intrinsic Reduced Nicotinamide Adenine Dinucleotide Fluorescence¶

“…Given that the cross section is measured in units of cm 4 s photon À1 , this specific dose parameter has units of photon 2 cm À4 s…”

“…Because BrdU incorporation is assayed by labeled DNA strand breaks, it is conceivable that photodamage resulting in such breaks could provide a false-positive indication of BrdU incorporation. There is a substantial literature indicating either direct or indirect strand breaks caused by intense near-infrared illumination of mammalian cells (see, for example, Mohanty et al [4]; Shafirovich et al [24] and references therein). Furthermore, cells spontaneously undergoing apoptosis could also lead to a false-positive artifact.…”