ABSTRACT
Highly confined DNA damage by femtosecond laser irradiation currently arises as a powerful tool to understand DNA repair in live cells as a function of space and time. However, the specificity with respect to damage type is limited. Here, we present an irradiation procedure based on a widely tunable Er/Yb : fiber femtosecond laser source that favors the formation of DNA strand breaks over that of UV photoproducts by more than one order of magnitude. We explain this selectivity with the different power dependence of the reactions generating strand breaks, mainly involving reactive radical intermediates, and the direct photochemical process leading to UV-photoproducts. Thus, localized multi-photon excitation with a wavelength longer than 1 microm allows for the selective production of DNA strand breaks at sub-micrometer spatial resolution in the absence of photosensitizers.
Subject(s)
DNA Breaks, Double-Stranded , Lasers , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Microscopy, Confocal , Photons , Reactive Oxygen Species/metabolism , Ultraviolet Rays , X-ray Repair Cross Complementing Protein 1ABSTRACT
Individual nanometer-sized plasmonic antennas are excited resonantly with few-cycle laser pulses in the near infrared. Intense third-harmonic emission of visible light prevails for fundamental photon energies below 1.1 eV. Interband luminescence and second harmonic generation occur solely at higher driving frequencies. We attribute these findings to multiphoton resonances with the d-band transitions of gold. The strong third-order signal allows direct measurement of a subcycle plasmon dephasing time of 2 fs, highlighting the efficient radiation coupling and broadband response of the devices.
ABSTRACT
The performance of a confocal microscopy setup based on a single femtosecond fiber system is explored over a broad range of pump wavelengths for both linear and nonlinear imaging techniques. First, the benefits of a laser source in linear fluorescence excitation that is continuously tunable over most of the visible spectrum are demonstrated. The influences of subpicosecond pulse durations on the bleaching behavior of typical fluorophores are discussed. We then utilize the tunable near-infrared output of the femtosecond system in connection with a specially designed prism compressor for dispersion control. Pulses as short as 33 fs are measured in the confocal region. As a consequence, 2 mW of average power are sufficient for two-photon microscopy in an organotypic sample from the mouse brain. This result shows great prospect for deep-tissue imaging in the optimum transparency window around 1100 nm. In a third experiment, we prove that our compact setup is powerful enough to exploit even higher-order nonlinearities such as three-photon absorption that we use to induce spatially localized photodamage in DNA.
Subject(s)
Microscopy, Confocal/methods , Microscopy, Fluorescence, Multiphoton/methods , Animals , Cattle , DNA Damage/radiation effects , Endothelial Cells/cytology , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Pyramidal Cells/cytologyABSTRACT
We report on a single-pass device that efficiently converts the broadband near-infrared output from a femtosecond fiber laser into a narrow spectrum in the visible. With fan-out poled MgO:LiNbO3 we obtain sub-picosecond, continuously tunable pulses in the 520-700 nm range. Conversion efficiencies as high as 30% are observed at typical pump power levels of 30 mW, corresponding to average output powers up to 9.5 mW. The specifications of our device are ideal for applications in confocal microscopy and frequency metrology.