Generation of DNA photolesions by two-photon absorption of a frequency-doubled Ti:sapphire laser

The formation of spatially localized regions of DNA damage by multiphoton absorption of light is an attractive tool for investigating DNA repair. Although this method has been applied in cells, little information is available about the formation of lesions by multiphoton absorption in the absence of exogenous or endogenous sensitizing agents. Therefore, we have investigated DNA damage induced in vitro by direct two-photon absorption of frequency-doubled femtosecond pulses from a Ti:sapphire laser. We first developed a quantitative polymerase chain reaction assay to measure DNA damage, and determined that the quantum yield of lesions formed by one-photon absorption of 254 nm light is 7.86 × 10−4. We then measured the yield of lesions resulting from exposure to the visible femtosecond laser pulses, which exhibited a quadratic intensity dependence. The two-photon absorption cross section of DNA has a value (per nucleotide) of 2.6 GM at 425 nm, 2.4 GM at 450 nm, and 1.9 GM at 475 nm. A comparison of these in vitro results to several in vivo studies of multiphoton photodamage indicates that the onset of DNA damage occurs at lower intensities in vivo; we suggest possible explanations for this discrepancy.