Manipulation of Single Cells with Sub-cellular Precision Using Femtosecond Laser Pulses

Femtosecond laser pulses centered at 800 nm are used to manipulate sub-cellular structures inside live and fixed cells. Using only a few nanojoules of laser pulse energy, we are able to selectively disrupt individual mitochondria in live bovine capillary epithelial (BCE) cells, and cleave single actin fibers in the cell cytoskeleton network of fixed human fibro-blast cells. We tightly focus femtosecond laser pulses using high numerical aperture (NA) microscope objectives to create high laser intensity in the sub-micrometer-sized focal volume. Laser energy is absorbed through non-linear mechanisms, vaporizing material only at the focus. Fluorescence microscopy studies of the photodisrupted cell samples show that cell structures outside the laser focus are unaffected. Furthermore, we use ethidium bromide, a nucleic-acid-binding fluorophore to test cell viability before and after laser irradiation. Live cells are impermeable to ethidium bromide. This agent only penetrate cells with compromised membranes, i.e. dead cells. Results show that target cells remain alive, with little fluorescence due to ethidium bromide, even after one or more mitochondria were disrupted. We therefore demonstrate the possibility of using femtosecond laser pulses for precise manipulation of sub-cellular structures and nano-scale surgery within live cells.