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Irradiance values employed in multiphoton microscopy are only one order of magnitude below the irradiance threshold for femtosecond optical breakdown in aqueous media (approximately equals 1.0 x 1013 W cm-2). At the breakdown threshold, a plasma with a free electron density of about 1021 cm-3 is generated, and the energy density in the breakdown region is sufficiently high to cause the formation of a bubble at the laser focus. We found previously that plasmas with a free electron density <1021 cm-3 are formed also in a fairly large irradiance range below the breakdown threshold. The present study investigates the chemical, thermal, and thermomechanical effects produced by these low-density plasmas, and their consequences for multiphoton microscopy. We use a rate equation model considering multiphoton ionization and avalanche ionization to numerically simulate the plasma formation. The value of the plasma energy density created by each laser pulse is then used to calculate the temperature distribution in the focal region. The results of the temperature calculations yield, finally, the starting point for calculations of the thermoelastic stresses that are generated during the formation of the low-density plasmas. We found that with femtosecond pulses a large 'tuning range' exists for the creation of spatially extremely confined chemical, thermal and mechanical effects via free electron generation through nonlinear absorption. Photochemical effects dominate at the lower end of this irradiance range, whereas at the upper end they are mixed with thermal effects and modified by thermoelastic stresses. Above the breakdown threshold, the spatial confinement is partly destroyed by cavitation bubble formation, and the laser-induced effects become more disruptive. Our simulations revealed that the highly localized ablation of subcellular structures recently demonstrated by other researchers are probably mediated by free-electron-induced chemical bond breaking and not related to heating or thermoelastic stresses. At the irradiance values employed in multiphoton microscopy (about 1/20 of the breakdown threshold), the model predicts, for (lambda) =800 nm wavelength, an electron density of about 1011 cm-3, sufficient to produce free electrons in the focal volume. Multiphoton microscopy may, hence, be accompanied by chemical effects arising from these electrons. We conclude that low density plasmas below the optical breakdown threshold can be a versatile tool for the manipulation of transparent biological media and other transparent materials but may also be a potential hazard in multiphoton microscopy and higher harmonic imaging.
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