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Proceedings Paper

Laser-mediated perforation of plant cells
Author(s): Martin Wehner; Philipp Jacobs; Dominik Esser; Helga Schinkel; Stefan Schillberg
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Paper Abstract

The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. Lasers are increasingly exploited for the manipulation of plant cells, enabling the study of biological processes on a subcellular scale including transformation to generate genetically modified plants. In our setup either a picosecond laser operating at 1064 nm wavelength or a continuous wave laser diode emitting at 405 nm are coupled into an inverse microscope. The beams are focused to a spot size of about 1.5 μm and the tobacco cell protoplasts are irradiated. Optoporation is achieved when targeting the laser focal spot at the outermost edge of the plasma membrane. In case of the picosecond laser a single pulse with energy of about 0.4 μJ was sufficient to perforate the plasma membrane enabling the uptake of dye or DNA from the surrounding medium into the cytosol. When the ultraviolet laser diode at a power level of 17 mW is employed an irradiation time of 200 - 500 milliseconds is necessary to enable the uptake of macromolecules. In the presence of an EYFP encoding plasmid with a C-terminal peroxisomal signal sequence in the surrounding medium transient transformation of tobacco protoplasts could be achieved in up to 2% of the optoporated cells. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.

Paper Details

Date Published: 13 July 2007
PDF: 9 pages
Proc. SPIE 6632, Therapeutic Laser Applications and Laser-Tissue Interactions III, 66321W (13 July 2007); doi: 10.1117/12.728113
Show Author Affiliations
Martin Wehner, Fraunhofer-Institute for Laser Technology (Germany)
Philipp Jacobs, Fraunhofer-Institute for Laser Technology (Germany)
Dominik Esser, Fraunhofer-Institute for Laser Technology (Germany)
Helga Schinkel, Fraunhofer-Institute for Molecular Biology and Applied Ecology (Germany)
Stefan Schillberg, Fraunhofer-Institute for Molecular Biology and Applied Ecology (Germany)

Published in SPIE Proceedings Vol. 6632:
Therapeutic Laser Applications and Laser-Tissue Interactions III
Alfred Vogel, Editor(s)

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