Ultrafast laser assisted microinjection enables distinct spatial localization pattern in cells and retina

Abstract

Laser microbeam has enabled highly precise non-contact delivery of exogenous materials into targeted cells, which has been a highly challenging task while using traditional methods without compromising cell viability. We report distinct spatial localization of impermeable substances into mammalian cells and goldfish retinal cells in explants subsequent to ultrafast laser microbeam assisted injection, realized by focusing a near infrared tunable Ti: sapphire laser beam. Introduction of impermeable dye into the cell through localized pore formation was confirmed by distinct fluorescence at the site of pore formation on the membrane and its spatiotemporal diffusion pattern through the nucleus. Indirect optoporation by bubble formation, external to cell, led to a similar spatial diffusion pattern but with a larger time constant for injection. Using optimized laser intensity, exposure and spatial irradiation pattern, desired spatial transfection patterns in goldfish retina explants were achieved as confirmed by expression of injected plasmids encoded for light-activable channelrhodopsin-2 (ChR2) ion channel tagged with fluorescent protein. Laser assisted delivery of exogenous material into specific area of three-dimensional neuronal tissue, such as the retina, will help to understand the functioning of neuronal circuitry of normal and degenerated retina.