The influence of the pulse length, tau , of ultrashort laser pulses at 780 and 920 nm on cell vitality and cellular reproduction has been studied. A total of 2400 nonlabeled cells were exposed to a highly focused scanning beam from a mode-locked 80-MHz Ti:sapphire laser with 60-micros pixel dwell time. For the same pulse energy, destructive effects were more pronounced for shorter pulses. The damage behavior was found to follow approximately a P(2)/tau dependence (P , mean power), indicating that cell destruction is likely based on a two-photon excitation process rather than a one- or a three-photon event. Therefore, femtosecond as well as picosecond pulses provide approximately the same relative optical window for safe two-photon fluorescence microscopy.

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We developed a nanotechnologyfor highly localized and ultraprecise non-contact laser surgery within living cells, in particular for nanoprocessing of intranuclear structures. We used this technology for precise knocking out of intracellular nanometer-sized regions within living cells. The highly localized destructive effects were based on nonlinear interactions between intense near infraredfemtosecond laser pulses with TW/cm2 light intensities and the biological target. The high intensity was achieved by diffraction limitedfocusing ofthe 30 m W laser beam ofa compact Titanium:Sapphire laser within the sub-femtoliter focal volume of a high numerical aperture objective. By means of dffraction-limited focusing and plasma-induced ablation, highly precise nanosurgery can be performed without apparent thermal or mechanical damage to surrounding intracellular areas. We used the laserfor precise non-invasive dissection ofchromosomes within a nucleus ofa living cell. Following laser treatment, the cell remained alive. The use ofnear infraredpulses provide the possibility ofnon-invasive intracellular nanoprocessing also within living tissue in depths ofmore than 100 microns. Current multiphoton laser scanning microscopes are used for non-destructive 3D fluorescence imaging of biomolecules'. However, above certain laser power thresholds destructive effects determine the laser beam -cell interaction2'3. At TW/cm2 light intensities, optical breakdown and plasma formation my occur. Our goal was to develop a nanotechnology for precise surgery within living cells based on plasma-induced ablation. We used the NIR beams of mode-locked Titanium:Sapphire lasers (76 MHz Mira model 900-F and 80 MHz Vitesse, Coherent, Santa Clara, USA) which were coupled to a modified inverted confocal laser scanning microscope (LSM4 10, Zeiss, Jena, Germany). A high numerical aperture objective (Plan Neofluar 40x, N.A. 1 .3 oil) was used to focus the beam to an aperture-diffraction limited sub-micron spot. The pulse duration at the sample was determined with an autocorrelator and found to be 170 fs. At a mean power of 30 mW, the peak power and the peak intensities reaches values of 2.4 kW and TW/cm2, respectively. This intensity was sufficient to evoke multiphoton induced optical breakdown and plasma formation. This allowed ultraprecise nonodissection of isolated human chromosomes with a resolution below 500 nm. We succeeded to use this technique to perform nonosurgery within the nucleus of a living round colcemide-exposed Chinese hamster ovary (CHO) cell.. For that purpose, we labeled nuclear DNA with the live cell fluorophore Hoechst 30042 and imaged the chromosomes by a two-photon fluorescence excitation process at 1 mW mean laser power. Subsequent single point exposure at 30 mW within the chosen chromosome resulted in precise chromosome dissection without damage to the nuclear envelope4. The cell remained alive. Nonlinear destructive effects of intense NIR femtosecond laser pulses can be used to knock out genomic regions within living cells. In pa...

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