Influence of laser parameters and staining on femtosecond laser-based intracellular nanosurgery

Femtosecond (fs) laser-based intracellular nanosurgery has become an important tool in cell biology, albeit the mechanisms in the so-called low-density plasma regime are largely unknown. Previous calculations of free-electron densities for intracellular surgery used water as a model substance for biological media and neglected the presence of dye and biomolecules. In addition, it is still unclear on which time scales free-electron and free-radical induced chemical effects take place in a cellular environment. Here, we present our experimental study on the influence of laser parameters and staining on the intracellular ablation threshold in the low-density plasma regime. We found that the ablation effect of fs laser pulse trains resulted from the accumulation of single-shot multiphoton-induced photochemical effects finished within a few nanoseconds. At the threshold, the number of applied pulses was inversely proportional to a higher order of the irradiance, depending on the laser repetition rate and wavelength. Furthermore, fluorescence staining of subcellular structures before surgery significantly decreased the ablation threshold. Based on our findings, we propose that dye molecules are the major source for providing seed electrons for the ionization cascade. Consequently, future calculations of free-electron densities for intracellular nanosurgery have to take them into account, especially in the calculations of multiphoton ionization rates.