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

AbstractTime-resolved imaging was used to examine the use of pulsed laser microbeam irradiation to produce cell lysis. Lysis was accomplished through the delivery of 6ns, λ=532nm laser pulses via a 40×, 0.8 NA objective to a location 10μm above confluent monolayers of PtK2 cells. The process dynamics were examined at cell surface densities of 600 and 1000cells/mm2 and pulse energies corresponding to 0.7×, 1×, 2×, and 3× the threshold for plasma formation. The cell lysis process was imaged at times of 0.5ns to 50μs 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 600ns 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 ∼60–85kPa for cell monolayers cultured at a density of 600cells/mm2 to ∼180–220kPa for a surface density of 1000cells/mm2. The implications for single cell lysis and microsurgery are discussed