Laser-based molecular delivery and its applications in plant science

Abstract

Lasers enable modification of living and non-living matter with submicron precision in a contact-free manner which has raised the interest of researchers for decades. Accordingly, laser technologies have drawn interest across disciplines. They have been established as a valuable tool to permeabilize cellular membranes for molecular delivery in a process termed photoinjection. Laser-based molecular delivery was first reported in 1984, when normal kidney cells were successfully transfected with a frequency-multiplied Nd:YAG laser. Due to the rapid development of optical technologies, far more sophisticated laser platforms have become available. In particular, near infrared femtosecond (NIR fs) laser sources enable an increasing progress of laser-based molecular delivery procedures and opened up multiple variations and applications of this technique.This review is intended to provide a plant science audience with the physical principles as well as the application potentials of laser-based molecular delivery. The historical origins and technical development of laser-based molecular delivery are summarized and the principle physical processes involved in these approaches and their implications for practical use are introduced. Successful cases of laser-based molecular delivery in plant science will be reviewed in detail, and the specific hurdles that plant materials pose will be discussed. Finally, we will give an outlook on current limitations and possible future applications of laser-based molecular delivery in the field of plant science.

Keywords: Laser transfection; Laser-tissue interaction; Optoporation; Photoinjection; Transformation.

Fig. 1

Sketch of a typical photoinjection experiment using an inverted microscopic setup and a pulsed laser source. The laser beam is focused onto the sample using an inverted microscope setup. A single laser pulse or a train of pulses facilitates of the cellular membrane and possibly the cell wall. The exact physical process of photoporation depends on the applied laser parameters and will be discussed in the following section. Plasmolyzing the plant cell prior to photoinjection supports the molecular uptake

Fig. 2

left: Overview comparing the time scales of different interaction and photopoinjection regimes. Note that the real values and borders vary largely on the respective conditions and can therefore only be regarded as rough estimates. Right: schematic depiction of the different interaction regimes. In the photomechanical regime, typically a single laser pulse with high energy (~ several 10 nJ for fs pulses) is applied, whereas the LDP requires multitudes of pulses with low energy (< 1 nJ) and high repetition rates (~ 80 MHz) to accumulate the photochemical effect. ${\tau }_D(water)$: thermal diffusion time in water for objective with high numerical aperture (NA), LDP: low-density plasma, NIR fs: near infrared femtosecond, CW: continious wave, ROS: reactive oxigen species

Fig. 3

high-speed imaging of a photoinjection event in the photodisruptive regime. The focal point is depicted by a white arrow in (a). The generation and progression of a short lived cavitaion bubble can be observed 4 µs (b) and 8 µs (c) after application of a single laser pulse. The differential images shown in the bottom row reflect the dynamics of the photoinjection. (image reprinted from Rukmana et al. [68])