Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform

Abstract

A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.

Biodegradable microsphere-mediated cell perforation in microfluidic channel using femtosecond laser

“…[1][2][3][4][5] Cell membrane perforation by focused femtosecond laser pulses provides an exclusive technology to introduce external molecules into a single cell, i.e., optoinjection, without significant cell damage because of a nonlinear interaction in a tiny focused spot. [6][7][8][9] Recently, the use of nanoparticles or microparticles has been accelerating attractive achievements to expand the capability of ultrashort laser optoinjection technology toward simultaneous treatment of multiple cells. Chakravarty et al 10 reported the delivery of DNA, protein, and fluorescence molecules into cells in a cuvette by using carbon black nanoparticles and a femtosecond laser.…”

Investigation of Biophysical Mechanisms in Gold Nanoparticle Mediated Laser Manipulation of Cells Using a Multimodal Holographic and Fluorescence Imaging Setup

“…The perforation efficiency was studied in a separate experiment. Propidium iodide was added before laser manipulation at a concentration of 1.5 μ M as also described by Antkowiak et al and Mitchell et al [ 19 , 33 ]. Here, propidium iodide enters the cell due to membrane perforation.…”

Enzyme-Assisted Photoinjection of Megadalton Molecules into Intact Plant Cells Using Femtosecond Laser Amplifier

“…More recently, a near infrared femtosecond (fs) laser has been employed for the nanoprocessing and photoinjection to plants 16–18 . One of the main advantages of the fs laser photoinjection is the precise perforation capability at a subcellular resolution thanks to effective non-linear processes such as a multi-photon absorption at a laser focal point, which suppresses collateral damage in the optical path.…”

“…Mitchell et al . 18 reported a systematic study of fs laser photoinjection efficiency of FITC-dextran into a tobacco BY-2 cell (TBY-2) considering the dependence on osmolarity, laser power and molecular weight of the conjugated dextran. They concluded that the introduction efficiency was significantly reduced with increasing molecular weight, and the maximum size of introduced dextran was 40 kDa under optimized conditions.…”