Details
| Original language | English |
|---|---|
| Article number | e79235 |
| Journal | PLoS ONE |
| Volume | 8 |
| Issue number | 11 |
| Publication status | Published - 14 Nov 2013 |
| Externally published | Yes |
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.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- General Biochemistry,Genetics and Molecular Biology
- Agricultural and Biological Sciences(all)
- General Agricultural and Biological Sciences
- General
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In: PLoS ONE, Vol. 8, No. 11, e79235, 14.11.2013.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Femtosecond optoinjection of intact tobacco BY-2 cells using a reconfigurable photoporation platform
AU - Mitchell, Claire A.
AU - Kalies, Stefan
AU - Cizmár, Tomás
AU - Heisterkamp, Alexander
AU - Torrance, Lesley
AU - Roberts, Alison G.
AU - Gunn-Moore, Frank J.
AU - Dholakia, Kishan
PY - 2013/11/14
Y1 - 2013/11/14
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84893643628&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0079235
DO - 10.1371/journal.pone.0079235
M3 - Article
C2 - 24244456
AN - SCOPUS:84893643628
VL - 8
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 11
M1 - e79235
ER -