We have shown by experimental investigations that cellular surgery (optoporation and optoinjection) with Nd:YAG laser pulses of 1064 nm and 532 nm wavelength relies on nonlinear absorption leading to optical breakdown and plasma formation at the laser focus.The present study explores the possibilities of refining the breakdown effects by making use of shorter pulse durations. Optical breakdown in water at large numerical aperture (NA = 0.9 and NA = 1.3) is simulated numerically for wavelengths of 1064 nm, 532 nm and 355 nm, and pulse durations of 6 ns, 30 ps and 100 fs. We use a rate equation model to calculate the temporal evolution of the free electron density during the laser pulse. Calculations are performed separately for the contributions of multiphoton ionization and avalanche ionization. We determine the maximum electron density reached during the laser pulse, the absorption coefficient of the plasma, and the volumetric energy density as a function of irradiance and wavelength. Experimental studies showed that self-focusing may be neglected for NA ³ 0.9.
The simulations predict that the energy threshold for cellular surgery at NA = 1.3 can be reduced by a factor of » 1000 when the pulse duration is reduced from 6 ns to 100 fs. The calculated breakdown energies for 100 fs pulses are about 1 nJ. With ns-pulses at 1064 nm, the breakdown threshold is very sharp, i. e. there is either no effect at all, or a dense plasma is formed causing a microexplosion. With shorter wavelengths and pulse durations, the threshold is smoother, and electron densities may be produced that stay below the threshold for explosive vaporization and bubble formation. This creates the possibility of achieving non-explosive plasma-mediated effects. Both the reduced energy threshold and the smoother breakdown process with fs pulses bear a large potential for the refinement of intracellular surgery.
The formation of low-density plasmas below the optical breakdown threshold together with the thermal effects arising from these plasmas were then investigated in more detail. Based on the data on the plasma energy density created by each laser pulse, the time evolution of the temperature distribution in the focal region is calculated for single laser pulses and series of pulse. 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 chemical and thermal effects via free electron generation through nonlinear absorption. Photochemical effects dominate at the lower end of this range, whereas at the upper end they are mixed with photothermal 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 intracellular structures and intranuclear chromosome dissection recently demonstrated by other researchers are probably mediated by free-electron-induced chemical bond breaking and not related to heating or thermoelastic stresses.
We conclude that low density plasmas below the optical breakdown threshold produced at large NA are a versatile tool for the manipulation of transparent biological media. Examples are intracellular surgery in living cells, knocking out of individual cell organelles for studies of their function, and isolation of individual cells or cell organelles for proteomic and genomic analysis. Low density plasmas may, however, also be a potential hazard in multiphoton microscopy and higher harmonic imaging in vivo.
Copyright. All Rights Reserved.