Pulsed Laser Microbeam-Induced Cell Lysis: Time-Resolved Imaging and Analysis of Hydrodynamic Effects

Abstract

Time-resolved imaging was used to examine the use of pulsed laser microbeam irradiation to produce cell lysis. Lysis was accomplished through the delivery of 6 ns, lambda=532 nm laser pulses via a 40x, 0.8 NA objective to a location 10 microm above confluent monolayers of PtK2 cells. The process dynamics were examined at cell surface densities of 600 and 1000 cells/mm2 and pulse energies corresponding to 0.7x, 1x, 2x, and 3x the threshold for plasma formation. The cell lysis process was imaged at times of 0.5 ns to 50 micros after laser pulse delivery and revealed the processes of plasma formation, pressure wave propagation, and cavitation bubble dynamics. Cavitation bubble expansion was the primary agent of cell lysis with the zone of lysed cells fully established within 600 ns of laser pulse delivery. The spatial extent of cell lysis increased with pulse energy but decreased with cell surface density. Hydrodynamic analysis indicated that cells subject to transient shear stresses in excess of a critical value were lysed while cells exposed to lower shear stresses remained adherent and viable. This critical shear stress is independent of laser pulse energy and varied from approximately 60-85 kPa for cell monolayers cultured at a density of 600 cells/mm2 to approximately 180-220 kPa for a surface density of 1000 cells/mm2. The implications for single cell lysis and microsurgery are discussed.

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

“…The radial dimension defining the zones of cellular injury as a function of pulse energy is shown in Table 1. As reported previously, we found the radius of cell lysis, R lys , to be 23, 30, 36, and 56μm for pulse energies corresponding to 1×, 2×, 3×, and 5× threshold, respectively [20], The viability assays revealed values of R necr to be 66, 105, 134, and 165μm for pulse energies corresponding to 1×, 2×, 3×, and 5× threshold, respectively. The maximum radial location occupied by permeabilized cells, R perm , were measured as 101, 162, 202, and 252μm for pulse energies corresponding to 1×, 2×, 3×, and 5× threshold, respectively.…”

“…Previously, we identified the radius of cell lysis R lys as the radius of the region around the irradiation site that was denuded of cells immediately after laser irradiation [20], Upon completion of the viability and membrane permeability assays, we found many cells beyond this region to be necrotic and, as mentioned previously and shown in Figure 2, detach during the wash process associated with the cell assays. Moreover, a few cells that remained adherent were also necrotic.…”

“…The threshold energy for plasma formation in our experimental system is 8μJ [20], This was verified through the delivery of a Nd:YAG laser pulse via the 40×, 0.8 NA bright-field objective into a Petri dish filled with culture medium. Plasma formation in the culture medium was observed visually in a dark room and its incidence for 50 pulses at discrete pulse energies was recorded.…”

“…As a result, there is an increasing interest to use pulsed laser microbeams for precise cellular manipulation, including laserinduced cell lysis [1], cell microdissection and catapulting [2][3][4][5], cell collection, expansion, and purification [6][7][8], cellular microsurgery [9][10][11], and cell membrane permeabilization for the delivery of membrane-impermeant molecules into cells [12 15], The processes of laser-induced optoinjection and optoporation offer the ability to load cells with a variety of biomolecules on short time scales (milliseconds to seconds) through optically produced cell membrane permeabilization [12,14,15], Despite the innovative utilization of laser microbeams in cell biology and biotechnology, only recently have studies provided insight regarding the mechanisms that mediate the interactions of highly focused pulsed laser beams with cells [16][17][18][19][20][21][22], A better understanding of these processes will prove critical to the continued development of laser microbeams for both research and practical applications. In previous studies, we provided a detailed characterization of the physics involved in the interaction of highly-focused nanosecond laser microbeams with cells [19,20], However, it is important to relate these physical effects to the biological response of the cells. The objective of this work is to determine the biological effects resulting from laser-induced cellular injury and connect these effects with the physics of the laser microbeam irradiation process.…”

“…Of particular interest is a hydrodynamic analysis of the cavitation bubble expansion that reveals the effects of fluid shear stress on cell lysis, viability, and transient membrane permeabilization for molecular delivery. We have recently utilized time-resolved imaging methods to show that the cavitation bubble expansion is the primary agent for the lysis of PtK 2 cells when using nanosecond laser microbeams focused at a location 10 μm above the cell monolayer [19,20], Cell lysis is initiated at the site of plasma formation and propagates outwards with the bubble expansion [20], A hydrodynamic model reveals that for cell monolayers cultured at surface densities of 1000 cells/mm 2 , cell lysis occurred only at locations where transient shear stresses τ w, max > 190 ± 20 kPa, independent of the laser pulse energy. Cells exposed to lower shear stresses remained intact and appeared viable even though they were subject to severe transient deformation during the cavitation bubble dynamics.…”