Optoperforation of single, intact Arabidopsis cells for uptake of extracellular dye-conjugated dextran

LeBlanc, Megan L.; Merritt, Travis R.; McMillan, Jameel; Westwood, James H.; Khodaparast, Giti A.

doi:10.1364/oe.21.014662

Cited by 8 publications

(8 citation statements)

References 20 publications

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“…The more severe the disruption to the membrane, the more likely that the cell will be non-viable post-irradiation as the membrane permeability may become permanently compromised. The maximum optoinjection efficiency achieved with either beam was over 10-fold higher than those presented previously applying a ns laser to intact BY-2 cells [17] and similar to those achieved using a femtosecond Gaussian beam in Arabidopsis epidermal cells [24] .…”

Section: Discussionsupporting

confidence: 80%

“…PI is membrane impermeable unless the cell membrane is compromised and it is used as a standard proof-of-concept photoporation fluorophore [44] – [46] . Upon entry into a photoporated cell, PI intercalates with nucleic acids present in the cytosol causing enhanced fluorescence [24] , which is seen experimentally in the cytoplasm. Optoinjection of PI into the cell can be seen in the fluorescent images from Figure 2 .…”

Section: Resultsmentioning

confidence: 94%

“…Fs optical injection and transfection has proven to be valuable for many different mammalian cell lines, particularly hard-to-transfect cell lines such as neurons [19] , [20] , stem cells [21] and in vivo systems [22] . With regard to plant cells, high-precision fs laser-mediated optoinjection of single cells within Arabidopsis root was reported first by Tirlapur and König [23] and has been investigated further in Arabidopsis epidermal cells [24] . While it is useful to explore single-cell photoporation in higher plant tissue, this cannot be considered particularly representative of the plant cell system due to the individual cells’ specialized states.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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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.

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Section: Discussionsupporting

confidence: 80%

Section: Resultsmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

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

et al. 2013

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“…The timescale from generation of quasi-free electrons, formation of ions and radicals (mainly derived from water molecules) and their recombination with biological molecules is in the range of nano- to microseconds and the effects accumulate over several laser pulses [ 12 , 60 – 62 ]. Since the plasma volume can be smaller than the actual optical focal volume, extremely precise processing below the optical resolution limit can be achieved [ 12 , 63 ]. Consequently, side effects should be lowest in this regime.…”

Section: Introduction and Theoretical Backgroundmentioning

confidence: 99%

Laser-based molecular delivery and its applications in plant science

et al. 2022

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Lasers enable modification of living and non-living matter with submicron precision in a contact-free manner which has raised the interest of researchers for decades. Accordingly, laser technologies have drawn interest across disciplines. They have been established as a valuable tool to permeabilize cellular membranes for molecular delivery in a process termed photoinjection. Laser-based molecular delivery was first reported in 1984, when normal kidney cells were successfully transfected with a frequency-multiplied Nd:YAG laser. Due to the rapid development of optical technologies, far more sophisticated laser platforms have become available. In particular, near infrared femtosecond (NIR fs) laser sources enable an increasing progress of laser-based molecular delivery procedures and opened up multiple variations and applications of this technique.This review is intended to provide a plant science audience with the physical principles as well as the application potentials of laser-based molecular delivery. The historical origins and technical development of laser-based molecular delivery are summarized and the principle physical processes involved in these approaches and their implications for practical use are introduced. Successful cases of laser-based molecular delivery in plant science will be reviewed in detail, and the specific hurdles that plant materials pose will be discussed. Finally, we will give an outlook on current limitations and possible future applications of laser-based molecular delivery in the field of plant science.

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“…Using fs laser photoinjection, LeBlanc et al . 17 introduced fluorescein isothiocyanate-conjugated dextran (FITC-dextran) of 10 kDa into an intact single cell of Arabidopsis thaliana . Mitchell et al .…”

Section: Introductionmentioning

confidence: 99%

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

Rukmana

Moran

Méallet‐Renault

et al. 2019

Sci Rep

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Femtosecond laser photoporation has become a popular method to deliver various kinds of molecules such as genes, proteins, and fluorescent dyes into single mammalian cells. However, this method is not easily applied to plant cells because their cell wall and turgor pressure prevent the delivery, especially for larger molecules than the mesh size of the cell wall. This work is the first demonstration of the efficient photoinjection of megadalton molecules into a cytoplasm of an intact single plant cell by employing a femtosecond laser amplifier under moderate enzyme treatment conditions. The intense femtosecond laser pulse effectively formed a pore on the cell wall and membrane of Tobacco BY-2, and 2 MDa dextran molecules were introduced through the pore. Along with the pore formation, induced mechanical tensile stresses on BY-2 cells were considered to increase permeability of the cell membrane and enhance the uptake of large molecules. Moreover, the moderate enzyme treatment partially degraded the cell wall thereby facilitating the increase of the molecular introduction efficiency.

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Optoperforation of single, intact Arabidopsis cells for uptake of extracellular dye-conjugated dextran

Cited by 8 publications

References 20 publications

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

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

Laser-based molecular delivery and its applications in plant science

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

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