Sub-cellular femtosecond laser ablation

We study the selective ablation by femtosecond laser pulses of sub-cellular structures in bovine endothelial cells, with selectively stained microtubules, actin fibers, and nuclei. The cells are placed in a custom-built inverted fluorescence microscope with a 1.4 NA oil-immersion objective. The laser used for ablation is centered at 800 nm delivering 100-fs laser pulses at a repetition rate of 1 kHz and the typical energy delivered at the sample is 1–5nJ. To determine the structural change and the size of the laser-affected area, we use transmission electron microscopy (TEM), in addition to standard fluorescence imaging. We also use the TEM images to discriminate laser-induced photobleaching of the fluorescent dye from material ablation, which appear identical in fluorescent imaging. The photobleaching time of the fluorescent dye is comparable to the laser processing time, making it difficult to observe long-term effects. To circumvent this problem we constructed a two-photon microscope using on a Coherent Mira femtosecond laser. The two-photon microscope allows long-time and high-spatial resolution observation.