Preserving the integrity of DNA is fundamental for cell survival. Therefore, DNA repair research has a high demand for methods to induce DNA damage with high spatial and temporal selectivity. To apply and study the effects of fs-laser irradiation in live cells, we have developed a multicolor fs-laser system. This turnkey system is based on single-mode fs-Er: and Yb:fiber laser technology. It synchronously provides pulses at 515 nm, 775 nm and 1035 nm wavelength with 40 MHz repetition rate. All three branches feature closely matched, bandwidth-limited pulse durations between 80 and 95 fs in the focus of a commercial laser-scanning microscope. An average optical output power from 80 to 2000 mW in the corresponding branches is provided. We apply a tandem scanning scheme in order to decouple nonlinear photomanipulation from conventional imaging.
We extensively analyzed the induction of DNA damage upon fs-laser irradiation via immunocytochemistry. A set of irradiation working conditions at 515 nm and 1035 nm has been identified that specifically induce either DNA strand breaks or UV-photoproducts. In subsequent live-cell experiments, we observed the generation of secondary breaks due to the activation of nucleotide-excision-repair at 515 nm wavelength irradiation. Such secondary reactions escape detection by conventional immunocytochemistry, but are revealed by our approach. Furthermore, we identified working conditions of irradiation at 775 nm driving two-photon-photoactivation of fluorescently labelled proteins within the nucleus without simultaneously triggering unwanted DNA lesions. We can therefore study the mobility of e.g. chromatin proteins at sites of DNA damage or perform functional cellular studies of mutant DNA repair proteins
Michael Schmalz, Ines Wieser, Felix Schindler, Martin Stoeckl, Alfred Leitenstorfer, and Elisa Ferrando-May, "Targeted control and analysis of DNA damage repair in human cells with a multicolor femtosecond fiber laser system (Conference Presentation)," Proc. SPIE 10492, Optical Interactions with Tissue and Cells XXIX, 1049202 (Presented at SPIE BiOS: January 29, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2291007.5751446491001.
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