Laser Microbeam-induced Spatiotemporal Change in Refractive Index of Chromosomes in Living PTK2 Cells

Abstract

Ultra-short laser pulses, which allow for confinement of damage to the focal volume, are used routinely to ablate organelles inside living cells, yet the physical mechanisms of this ablation are not completely understood. Many different parameters, particularly wavelength and laser pulse duration, are used in experiments from different laboratories, but no systematic delineation of threshold energy and irradiance for microsurgery of chromosomes using various wavelengths has been reported.In a series of comparative experiments using three different lasers - a 200 femtosecond near-infrared, a 12 picosecond green, and a 12 nanosecond green - we measured energy and irradiance thresholds for inducing a change in the refractive index of micro-irradiated regions of chromosomes in mitotic PTK2 (Potorous tridactylus) kidney cells. To determine the exact irradiance at the focal point, we used the dual objective method for the wavelengths used in our study. While the required energy per pulse for a detectable change in refractive index was highest for the nanosecond laser, the required peak irradiance was highest for the femtosecond laser. The width of the observed damaged spot varied with pulse duration. The threshold for this change in refractive index was found to depend on the spatial pattern of irradiation - point versus line. Further, for a fixed wavelength, the time required to induce a detectable change in the refractive index decreased with increasing exposure time.In addition, a quantitative phase imaging system to quantify the damage in the micro-irradiated regions is being developed. This system will allow more precise quantitation of the change in refractive index in the chromosomes following exposure to the different laser parameters.From these results, specific insights can be gained into the physical mechanisms of laser ablation.