Generation of multiple DNA lesions at subnuclear resolution by multi-photon irradiation

Abstract

In the field of DNA repair, multi-photon absorption is becoming increasingly popular as a tool to induce DNA damage with high spatial resolution. Typically, cell nuclei of live cells are subjected to a defined pattern of irradiation at a wavelength around 800 nm, which is the characteristic output of femtosecond Ti:Sapphire lasers. This allows analyzing the spatial and temporal aspects of the recruitment of nuclear proteins, in particular repair factors, at the site of damage. Various types of DNA lesions, like UV-photoproducts [1], strand breaks [2] and reactive oxygen species [3, 4] were reported by different studies using femtosecond pulses with comparable parameters. This method thus appears to lack specificity with respect to the type of DNA damage inflicted [5] hampering the analysis of individual DNA repair pathways. In this study we take advantage of a tunable Er:fiber laser source [6] to investigate DNA damage induction by multiphoton absorption at λ > 800 nm and assess its specificity. To this end, we have increased the output power of our system by integrating a homebuilt Yb:fiber amplifier and a subsequent grating compressor. The output of the Yb-amplifier has a center wavelength of 1050 nm with a full width half maximum (FWHM) of 50 nm, the pulse duration in the focal plane of the objective-lens is sub-100 fs and the average power after the grating compressor is 250 mW. We present a comparative analysis of DNA-damage induced by multi-photon absorption at 774 nm (Figure 1.) and 1050 nm based on damage-specific immunostainings and the recruitment of various DNA repair factors. Potential mechanisms underlying the difference in the observed spectrum of lesions will be discussed.