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Nanojoule and sub-nanojoule 80 MHz femtosecond laser pulses at 750-850 nm of a compact titanium:sapphire laser have been used for highly precise nanoprocessing of DNA as well as of intracellular and intratissue compartments. In particular, a mean power between 15 mW and 100 mW, 170 fs pulse width, submicron distance of illumination spots and microsecond beam dwell times on spots have been used for multiphoton- mediated nanoprocessing of human chromosomes, brain and ocular intrastromal tissue. By focusing the laser beam with high numerical aperture focusing optics of the laser scan system femt-O-cut and of modified multiphoton scanning microscopes to diffraction-limited spots and TW/cm2 light intensities, precise submicron holes and cuts have been processed by single spot exposure and line scans. A minimum FWHM cut size below 70 nm during the partial dissection of the human chromosome 3 was achieved. Complete chromosome dissection could be performed with FWHM cut sizes below 200 nm. Intracellular chromosome dissection was possible. Intratissue processing in depths of 50 - 100micrometers and deeper with a precision of about 1micrometers including cuts through a nuclei of a single intratissue cell without destructive photo-disruption effects to surrounding tissue layers have been demonstrated in brain and eye tissues. The femt-O-cut system includes a diagnostic system for optical tomography with submicron resolution based on multiphoton- excited autofluorescence imaging (MAI) and second harmonic generation. This system was used to localize the intracellular and intratissue targets and to control the effects of nanoprocessing. These studies show, that in contrast to conventional approaches of material processing with amplified femtosecond laser systems and (mu) J pulse energies, nanoprocessing of materials including biotissues can be performed with nJ and sub-nJ high repetition femtosecond laser pulses of turn-key compact lasers without collateral damage. Potential applications include highly precise cell and embryo surgery, gene diagnostics and gene therapy, intrastromal refractive surgery, cancer therapy and brain surgery.
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