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Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform
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-impe...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3828288/ https://www.ncbi.nlm.nih.gov/pubmed/24244456 http://dx.doi.org/10.1371/journal.pone.0079235 |
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author | Mitchell, Claire A. Kalies, Stefan Cizmár, Tomás Heisterkamp, Alexander Torrance, Lesley Roberts, Alison G. Gunn-Moore, Frank J. Dholakia, Kishan |
author_facet | Mitchell, Claire A. Kalies, Stefan Cizmár, Tomás Heisterkamp, Alexander Torrance, Lesley Roberts, Alison G. Gunn-Moore, Frank J. Dholakia, Kishan |
author_sort | Mitchell, Claire A. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3828288 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-38282882013-11-16 Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform Mitchell, Claire A. Kalies, Stefan Cizmár, Tomás Heisterkamp, Alexander Torrance, Lesley Roberts, Alison G. Gunn-Moore, Frank J. Dholakia, Kishan PLoS One Research Article 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. Public Library of Science 2013-11-14 /pmc/articles/PMC3828288/ /pubmed/24244456 http://dx.doi.org/10.1371/journal.pone.0079235 Text en © 2013 Mitchell et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Mitchell, Claire A. Kalies, Stefan Cizmár, Tomás Heisterkamp, Alexander Torrance, Lesley Roberts, Alison G. Gunn-Moore, Frank J. Dholakia, Kishan Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform |
title | Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform |
title_full | Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform |
title_fullStr | Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform |
title_full_unstemmed | Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform |
title_short | Femtosecond Optoinjection of Intact Tobacco BY-2 Cells Using a Reconfigurable Photoporation Platform |
title_sort | femtosecond optoinjection of intact tobacco by-2 cells using a reconfigurable photoporation platform |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3828288/ https://www.ncbi.nlm.nih.gov/pubmed/24244456 http://dx.doi.org/10.1371/journal.pone.0079235 |
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