Abstract
A plant science research goal is to manipulate single cells in an intact organism in order to study their interactions with neighboring cells. Based on a technique previously demonstrated in isolated plant cells, mammalian cells and cyanobacteria, Arabidopsis epidermal cells were optoperforated to allow for uptake of external cascade blue-labeled dextrans. Adverse organelle responses were determined to be minimal and dye retention was demonstrated for at least 72 hours. This technique overcomes the physical challenges presented by the plant cell wall and demonstrates the feasibility of in situ optoperforation.
Highlights
Optoperforation (OP) can be found in scientific literature under many names: nanoinjection, optical injection, phototransfection, photoporation, or micropuncture; all of which refer to the technique of using lasers to form a quasi-free electron plasma at a cellular boundary to increase its permeability, allowing uptake of materials from a solution surrounding the cell [1]
To explain the mechanisms responsible for femtosecond laser optoperforation, we rely on the theoretical framework proposed by Vogel et al [15]
Since such requisite intensities are only found within the focal volume, the generated quasi-free electron plasma is relegated within this volume
Summary
Optoperforation (OP) can be found in scientific literature under many names: nanoinjection, optical injection, phototransfection, photoporation, or micropuncture; all of which refer to the technique of using lasers to form a quasi-free electron plasma at a cellular boundary to increase its permeability, allowing uptake of materials from a solution surrounding the cell [1]. UV-laser irradiated Brassica napus cultures in hypertonic solution with a β-glucuronidase (gusA) expressing construct had a 50% survivability rate of perforated cells two days post laser exposure and gusA expression was detected in 71% of survivors after 4 days [3]. Cultured cells of Brassica napus have been shown to remain viable following UV (343 nm, 15 ns) laser irradiation followed by uptake of bizbenzimide-labelled pBR322 plasmid, with cytoplasmic streaming visible one hour post treatment and similar survival rates [3]. Cultured wheat cells were transformed with a gusA expression vector by a UV laser (6 ns, 308 nm with pulse energies up to 13 mJ) with confirmation of transformation seen in 3 out of 600 samples [4]
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