Biophysical Response to Pulsed Laser Microbeam‐Induced Cell Lysis and Molecular Delivery

Abstract

Cell lysis and molecular delivery in confluent monolayers of PtK 2 cells are achieved by the delivery of 6 ns, λ = 532 nm laser pulses via a 40×, 0.8 NA microscope objective. With increasing distance from the point of laser focus we find regions of (a) immediate cell lysis; (b) necrotic cells that detach during the fluorescence assays; (c) permeabilized cells sufficient to facilitate the uptake of small (3kDa) FITC-conjugated Dextran molecules in viable cells; and (d) unaffected, viable cells. The spatial extent of cell lysis, cell detachment, and molecular delivery increased with laser pulse energy. Hydrodynamic analysis from time-resolved imaging studies reveal that the maximum wall shear stress associated with the pulsed laser microbeam-induced cavitation bubble expansion governs the location and spatial extent of each of these regions independent of laser pulse energy. Specifically, cells exposed to maximum wall shear stresses τ w, max > 190 ± 20 kPa are immediately lysed while cells exposed to τ w, max > 18 ± 2kPa are necrotic and subsequently detach. Cells exposed to τ w, max in the range 8-18 kPa are viable and successfully optoporated with 3kDa Dextran molecules. Cells exposed to τ w, max < 8 ± 1 kPa remain viable without molecular delivery. These findings provide the first direct correlation between pulsed laser microbeam-induced shear stresses and subsequent cellular outcome.

Low-density plasma formation in aqueous biological media using sub-nanosecond laser pulses

“…9,12 While the delivery of nanosecond pulses at low NA may provide a less costly and more reproducible means to perform cellular manipulation at high throughput, current studies indicate that nanosecond optical breakdown is typically associated with large cavitation bubbles and extended (>100 lm) regions of cellular damage, even when using high numerical apertures. 13,14 This is consistent with experimental studies demonstrating that the high energies/irradiances required to initiate optical breakdown using nanosecond pulses inevitably generates vigorous avalanche ionization leading to the formation of a highly ionized plasma. 5,15 Interestingly, even though the mechanism is uncertain, reports have documented the effective use of subnanosecond pulses, delivered at low numerical aperture and scanned over large areas, to provide cell lysis and molecular delivery with high throughput and precision.…”

Dynamics of laser-induced bubble pairs

“…, Hellman et al (2008) and Compton, Hellman & Venugopalan (2013) have analysed the action of shear forces induced by individual laser-produced cavitation bubbles on groups of cells. Laser pulses with energies of a few microjoules (Hellman et al 2008;Compton et al 2013) or millijoules were focused onto an adherent cell layer in a Petri dish, and the flow and shear forces created by the resulting oscillating bubble was used for cell poration. In the immediate vicinity of the plasma, all cells were destroyed, but within a certain distance range from the breakdown site, shear forces were appropriate to cause membrane permeabilization on vital cells.…”

Spatially optimized gene transfection by laser-induced breakdown of optically trapped nanoparticles

“…This is typically an order of magnitude smaller than for previously reported studies 12,13 which utilized the breakdown of the surrounding buffer medium in which cells are cultured. Furthermore, we parallelized the process to allow simultaneous multisite targeting nanosurgery of cells using a spatial light modulator ͑SLM͒ for multipoint nanoparticle trapping as well as the multiplexing of the nanosecond laser beam for LIB of individual nanoparticles located in a spatially extended array of optical traps.…”

“…This results in a much larger cavitation bubble compared to the cell size that effectively reduces cell viability. [11][12][13] In this letter, we demonstrate targeted membrane permeabilization of multiple cells within a specific and controllable area utilizing cavitation bubbles excited by the laser-induced breakdown ͑LIB͒ of one or more optically trapped nanoparticles. When light from a nanosecond pulsed laser is focused onto an optically trapped nanoparticle, LIB can take place, 14 leading to the formation of plasma and emission of shockwaves by its expansion followed by the vaporization of the nanoparticle or liquid ͑surrounding aqueous medium͒.…”