Femtosecond laser nano-ablation in fixed and non-fixed cultured cells.

To understand the onset and morphology of femtosecond laser submicron ablation in cells and to study physical evidence of intracellular laser irradiation, we used transmission electron microscopy (TEM). The use of partial fixation before laser irradiation provides for clear images of sub-micron intracellular laser ablation, and we observed clear evidence of bubble-type physical changes induced by femtosecond laser irradiation at pulse energies as low as 0.48 nJ in the nucleus and cytoplasm. By taking ultrathin sliced sections, we reconstructed the laser affected subcellular region, and found it to be comparable to the point spread function of the laser irradiation. Laser-induced bubbles were observed to be confined by the surrounding intracellular structure, and bubbles were only observed with the use of partial pre-fixation. Without partial pre-fixation, laser irradiation of the nucleus was found to produce observable aggregation of nanoscale electron dense material, while irradiation of cytosolic regions produced swollen mitochondria but residual local physical effects were not observed. This was attributed to the rapid collapse of bubbles and/or the diffusion of any observable physical effects from the irradiation site following the laser exposure.