In a series of elegant experiments, Kuetymeyer et al. investigate the mechanism for dye-induced decrease in ablation threshold, as well as determine how varying laser parameters affects the ablation threshold. A microscope–laser system was used that allowed regulation of wavelength (680–1080 nm), pulse frequency (1 kHz – 80 MHz), and laser beam scanning capabilities. Tissue ablation was determined by the ability of the tissue to retain Hoechst 33342 (a nuclear stain) post-irradiation. The repetition rate of the laser was varied at a constant wavelength, and the scan speed was adjusted to keep the radiant energy density constant. It was found that the exposure time per micrometer was inversely proportional to the fourth power of irradiance. From these results Kuetymeyer et al. draw three conclusions: (1) minimal thermal damage occurs, (2) tissue ablation occurs from the accumulation of photochemical damage from each laser pulse, and (3) multiphoton absorption is required as the precursor for the photochemical damage. By studying the ablation threshold in the absence and presence of Hoechst 33342, it was found that the Hoechst dye lowered the ablation threshold by a factor of 4. In Hoechst-stained cells, the ablation threshold was monitored in a single spot while the wavelength of the laser was varied and the repetition rate was kept fixed (kilohertz regime). It was determined that the number of electrons required for ablation was increased from 4 to 5 as the wavelength increased from 840 to 950 nm, corresponding to 5.2–5.9 eV ionization energy and indicating the Hoechst dye as the seed for electrons, not water. In a final experiment, the full-width at half maximum (FWHM) of the damaged/ablated regions was studied as a function of pulse energy and repetition rate. It was found that the repetition rate had no effect on the ablated regions when pulse energies were varied up to 4 Mhz but that a significant change in the FWHM of the damaged region was seen when the repetition rate was increased to 80 MHz. The increase in ablation area indicates that more seed electrons are produced with high repetition rates. This work shows that there are two ways to minimize the total laser energy delivered in intracellular nanosurgery: (1) utilize a dye capable of providing seed electrons and (2) use high laser repetition rates between 700 and 800 nm.
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