Tissue surgery and subcellular photodisruption with femtosecond laser pulses

Abstract:

The short duration of femtosecond laser pulses allows high laser intensities to be reached with low pulse energies. Focusing pulses through high numerical aperture microscope objectives leads to intensities high enough to induce plasma formation and photodisruption of matter at the laser focus through nonlinear mechanisms. These studies investigate the potential for using femtosecond lasers for photodisruptive surgery on the surface and in the bulk of turbid tissue. As our tissue model, we use mouse skin tissue. Our experiments demonstrate that incisions are made on the tissue surface by translating the laser focus across the sample while irradiating with a continuous train of pulses. Subsurface cavities are made in the tissue bulk by focusing the laser beneath the surface. Images of histological sections of samples show that damage microstructures are created with high precision and minimal collateral damage outside the focal region. We find that there is a maximum depth at which subsurface cavities can be made; placing the laser focus below this depth results in filament formation through the nonlinear optical phenomenon of selffocusing. Studies of photodisruption in fixed cell samples show that damage morphologies are made on the size scale of subcellular structures. We demonstrate that cells can be fluorescently labeled for specific cellular structures and that photodisruption of these samples can be effectively discerned from photobleaching effects. Subcellular damage is also shown to be localized completely within the cell. We demonstrate the feasibility of using femtosecond laser pulses for manipulation of subcellular structures in living cells, and we show that this can be done without compromising cell membrane integrity.