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Hydrodynamic determinants of cell necrosis and molecular delivery produced by pulsed laser microbeam irradiation of adherent cells.

ABSTRACT: Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at ? = 532 nm was delivered to confluent monolayers of PtK2 cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180-1100 ps and pulse energies of 0.5-10.5 ?J, we examined the resulting plasma formation and cavitation bubble dynamics that lead to laser-induced cell lysis, necrosis, and molecular delivery. The cavitation bubble dynamics are imaged at times of 0.5 ns to 50 ?s after the pulsed laser microbeam irradiation, and fluorescence assays assess the resulting cell viability and molecular delivery of 3 kDa dextran molecules. Reductions in both the threshold laser microbeam pulse energy for plasma formation and the cavitation bubble energy are observed with decreasing pulse duration. These energy reductions provide for increased precision of laser-based cellular manipulation including cell lysis, cell necrosis, and molecular delivery. Hydrodynamic analysis reveals critical values for the shear-stress impulse generated by the cavitation bubble dynamics governs the location and spatial extent of cell necrosis and molecular delivery independent of pulse duration and pulse energy. Specifically, cellular exposure to a shear-stress impulse J?0.1 Pa s ensures cell lysis or necrosis, whereas exposures in the range of 0.035?J?0.1 Pa s preserve cell viability while also enabling molecular delivery of 3 kDa dextran. Exposure to shear-stress impulses of J?0.035 Pa s leaves the cells unaffected. Hydrodynamic analysis of these data, combined with data from studies of 6 ns microbeam irradiation, demonstrates the primacy of shear-stress impulse in determining cellular outcome resulting from pulsed laser microbeam irradiation spanning a nearly two-orders-of-magnitude range of pulse energy and pulse duration. These results provide a mechanistic foundation and design strategy applicable to a broad range of laser-based cellular manipulation procedures.

SUBMITTER: Compton JL

PROVIDER: S-EPMC3824719 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Hydrodynamic determinants of cell necrosis and molecular delivery produced by pulsed laser microbeam irradiation of adherent cells.

Compton Jonathan L JL Hellman Amy N AN Venugopalan Vasan V

Biophysical journal 20131101 9

Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at λ = 532 nm was delivered to confluent monolayers of PtK2 cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180-1100 ps and pulse energies of 0.5-10.5 μJ, we examined the resulting plasma formation a ...[more]

PMID: 24209868

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Project description:Laser pulse processing of surfaces and thin films is a useful tool for amorphous thin films crystallization, surface nanostructuring, phase transformation and modification of physical properties of thin films. Here we show the effects of nanostructuring produced at the surface and under the surface of amorphous GeTiO films through laser pulses using fluences of 10-30 mJ/cm(2). The GeTiO films were obtained by RF magnetron sputtering with 50:50 initial atomic ratio of Ge:TiO2. Laser irradiation was performed by using the fourth harmonic (266 nm) of a Nd:YAG laser. The laser-induced nanostructuring results in two effects, the first one is the appearance of a wave-like topography at the film surface, with a periodicity of 200 nm and the second one is the structure modification of a layer under the film surface, at a depth that is related to the absorption length of the laser radiation. The periodicity of the wave-like relief is smaller than the laser wavelength. In the modified layer, the Ge atoms are segregated in spherical amorphous nanoparticles as a result of the fast diffusion of Ge atoms in the amorphous GeTiO matrix. The temperature estimation of the film surface during the laser pulses shows a maximum of about 500 °C, which is much lower than the melting temperature of the GeTiO matrix. GeO gas is formed at laser fluences higher than 20 mJ/cm(2) and produces nanovoids in the laser-modified layer at the film surface. A glass transition at low temperatures could happen in the amorphous GeTiO film, which explains the formation of the wave-like topography. The very high Ge diffusivity during the laser pulse action, which is characteristic for liquids, cannot be reached in a viscous matrix. Our experiments show that the diffusivity of atomic and molecular species such as Ge and GeO is very much enhanced in the presence of the laser pulse field. Consequently, the fast diffusion drives the formation of amorphous Ge nanoparticles through the segregation of Ge atoms in the GeTiO matrix. The nanostructuring effects induced by the laser irradiation can be used in functionalizing the surface of the films.

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Hydrodynamic determinants of cell necrosis and molecular delivery produced by pulsed laser microbeam irradiation of adherent cells.

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Hydrodynamic determinants of cell necrosis and molecular delivery produced by pulsed laser microbeam irradiation of adherent cells.

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