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Highly focused laser microbeams are being used with increasing regularity for targeted cell lysis, cellular microsurgery and molecular delivery via transient cell membrane permeabilization. To examine the mechanisms of laser induced cell lysis, we performed time-resolved imaging of confluent PtK2 cell cultures following the delivery of a single 6 ns, 532 nm Nd:YAG laser pulse. The laser pulse energies employed correspond to 1x and 3x threshold for plasma formation. The resulting plasma formation, pressure wave propagation and cavitation bubble dynamics were imaged over a temporal range spanning 5 orders of magnitude (0.5 ns - 50 µs). Time-resolved imaging enabled determination of process characteristics including pressure wave speed and amplitude and cavitation bubble energies. The time-resolved images also revealed the onset of cellular damage to occur on nano-second time scales and complete within 1 µs. Moreover, the size of the damage zone was larger than the plasma but smaller than the maximum cavitation bubble size. This indicated that mechanisms apart from plasma vaporization namely pressure wave propagation and cavitation bubble expansion are contributors to cellular damage. Dye exclusion assays showed that the majority of cells experiencing considerable deformation due to fluid flow generated by the cavitation bubble expansion remain viable over 24 hours.

Examination of laser-induced cell lysis by time resolved imaging