title: Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.
abstract: Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments.
authors.affiliation: Department of Biology, Bioimaging Center and Center for Applied Photonics, University of Konstanz Konstanz, Germany.
authors.forename: Elisa
authors.initials: E
authors.lastname: Ferrando-May
authors.forename: Martin
authors.initials: M
authors.lastname: Tomas
authors.forename: Philipp
authors.initials: P
authors.lastname: Blumhardt
authors.forename: Martin
authors.initials: M
authors.lastname: Stöckl
authors.forename: Matthias
authors.initials: M
authors.lastname: Fuchs
authors.forename: Alfred
authors.initials: A
authors.lastname: Leitenstorfer
keywords: DNA strand break; fluorescence; microirradiation; non-linear optics