High-throughput optical injection of mammalian cells using a Bessel light beam

Rendall, Helen Anne; Marchington, Robert F.; Praveen, Bavishna B.; Bergmann, G.; Arita, Yoshihiko; Heisterkamp, Alexander; Gunn-Moore, Frank J.; Dholakia, Kishan

doi:10.1039/c2lc40708f

Cited by 33 publications

(23 citation statements)

References 21 publications

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“…In previous studies (1)(2)(3)(4)(5)(6)(7)(8), researchers optoporated cells using high-repetition rate trains (~80 MHz) of lowenergy (~1 nJ) near-infrared (800 nm) laser pulses that were focused at high numerical aperture (0.8-1.4 NA), or directed Bessel beams (9) to the membrane of targeted cells for short durations (4-250 ms). In this regime, nonlinear absorption of laser energy in the focal volume is thought to lead to the production of a low-density electron plasma that causes disruption of chemical bonds, release of free electrons, and (potentially) localized heating (10).…”

Section: Introductionmentioning

confidence: 99%

Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser Pulses

Davis

Farrar

Nishimura

et al. 2013

Biophysical Journal

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Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irradiation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell transfection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggesting that plasmids may enter the cell by adhering to the membrane and then being translocated.

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Section: Introductionmentioning

confidence: 99%

Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser Pulses

Davis

Farrar

Nishimura

et al. 2013

Biophysical Journal

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“…In addition the applied mode of action only allows manipulation of single cells as the laser focus has to be carefully aligned to the membrane of each cell. Lately, several approaches have tried to overcome this drawback by the use of non-diffractive laser beams [7], micro fluidic channels [8] or implementation of a spatial light modulator to create multiple foci [9]. So far, none of these approaches can provide an efficient, fast and intuitively usable transfection platform.…”

Section: Introductionmentioning

confidence: 99%

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

et al. 2013

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Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.

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“…However, each cell has to be porated individually by focusing the laser beam directly onto the membrane, causing optoporation to have an extremely low throughput. Modifications, including the use of active flow in microfluidic channels and a nondiffracting beam, slightly increase the throughput but not to the scale necessary for applications such as cell therapy, which can require on the order of 10 8 cells [35,36].…”

Section: Laser-mediated Cell Poration For Intracellular Deliverymentioning

confidence: 99%

Plasmonic Intracellular Delivery

Madrid¹

2018

Plasmonics

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This chapter describes the significance of plasmonics to the field of intracellular delivery. We begin by discussing the significance of intracellular delivery, its applications in biology and medicine, and the currently available intracellular delivery techniques. Next, we discuss the field of plasmonic intracellular delivery, beginning with the discovery of optoporation. In optoporation, a laser beam is tightly focused onto a cell membrane to generate a transient pore, through which membrane-impermeable cargo can enter the cell. To improve the throughput of this technique, plasmonic materials were used for their ability to efficiently absorb laser light and generate spatially confined electric fields. Here, we describe the process by which plasmonic materials absorb laser light energy and generate plasmons. These plasmons transfer their energy to their surroundings, resulting in a rise in temperature and the subsequent creation of a bubble or shockwave. Finally, we describe how the properties of plasmons and plasmon-mediated effects facilitate cell poration for intracellular delivery.

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High-throughput optical injection of mammalian cells using a Bessel light beam

Cited by 33 publications

References 21 publications

Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser Pulses

Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser Pulses

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

Plasmonic Intracellular Delivery

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