Repetition rate dependency of reactive oxygen species formation during femtosecond laser–based cell surgery

Abstract

Femtosecond (fs) laser-based cell surgery is typically done in two different regimes, at kHz or MHz repetition rate. Formation of reactive oxygen species (ROS) is an often predicted effect due to illumination with short laser pulses in biological tissue. We present our study on ROS formation in single cells in response to irradiation with fs laser pulses depending on the repetition rate while focusing into the cell nucleus. We observed a significant increase of ROS concentration directly after manipulation followed by a decrease in both regimes at kHz and MHz repetition rate. In addition, effects of consecutive exposures at MHz and kHz repetition rate and vice versa on ROS production were studied. Irradiation with a MHz pulse train followed by a kHz pulse train resulted in a significantly higher increase of ROS concentration than in the reversed case and often caused cell death. In the presence of the antioxidant ascorbic acid, accumulation of ROS and cell death were strongly reduced. Therefore, addition of antioxidants during fs laser-based cell surgery experiments could be advantageous in terms of suppressing photochemical damage to the cell.

Mechanisms of nanoparticle-mediated photomechanical cell damage

“…Membrane damage might be supported by repetitive stress from a number of small vapor bubbles or be induced via biochemical effects such as oxidative stress from generation of reactive oxygen species or release of intracellular Ca 2+ . Such biochemical effects have been reported for ultrasound exposure [37] and fs-pulse irradiation [38,39] and may occur upon exposure to ns-pulse induced vapor bubble formation in the immediate vicinity of cell organelles. However, we believe that the most important reason for the lower damage threshold under multiple pulse exposure is related to the change in position of the nanoparticles.…”

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

“…Nikogosyan et al showed that photochemical processes leading to generation of ROS in aqueous solution can be classified into fast and slow reactions with time constants in the nano- to microsecond and millisecond range, respectively [21]. Recent experimental studies by our group revealed an influence of the laser repetition rate on the efficiency of ROS formation in cells and tissue [25, 26]. Consequently, the time interval between two successive laser pulses may affect the ablation threshold of subcellular structures.…”

Monitoring of laser micromanipulated optically trapped cells by digital holographic microscopy

“…Fluorescence methods already have been successfully utilized for confirming the optical transfection of cells (see e.g. [15][16][17]) but this requires a cell modification or sensing with specific dyes which may be toxic. Furthermore, a maximized performance of fluorescence imaging needs a precise focussing of the sample which becomes in particular a challenge in combination with multiple optical tweezers and microfluidics.…”