Rau Kaustubh R, Quinto-Su Pedro A, Hellman Amy N, Venugopalan Vasan
Laser Microbeam and Medical Program, Beckman Laser Institute, Department of Biomedical Engineering, University of California, Irvine, California, USA.
Biophys J. 2006 Jul 1;91(1):317-29. doi: 10.1529/biophysj.105.079921. Epub 2006 Apr 14.
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.
采用时间分辨成像技术来研究利用脉冲激光微束辐照产生细胞裂解的情况。通过一个40倍、数值孔径为0.8的物镜，将波长λ = 532 nm、脉宽6 ns的激光脉冲传输至汇合的PtK2细胞单层上方10微米处，从而实现细胞裂解。在细胞表面密度为600个细胞/mm²和1000个细胞/mm²，以及对应于等离子体形成阈值的0.7倍、1倍、2倍和3倍的脉冲能量条件下，对该过程的动力学进行了研究。在激光脉冲传输后的0.5 ns至50 μs时间段内，对细胞裂解过程进行成像，揭示了等离子体形成、压力波传播和空化泡动力学过程。空化泡膨胀是细胞裂解的主要因素，在激光脉冲传输后的600 ns内，裂解细胞区域完全形成。细胞裂解的空间范围随脉冲能量增加而增大，但随细胞表面密度降低。流体动力学分析表明，受到超过临界值的瞬态剪应力作用的细胞会被裂解，而受到较低剪应力作用的细胞则保持附着且存活。这种临界剪应力与激光脉冲能量无关，对于密度为600个细胞/mm²培养的细胞单层，其值约为60 - 85 kPa，对于表面密度为1000个细胞/mm²的情况，其值约为180 - 220 kPa。文中还讨论了对单细胞裂解和显微手术的意义。
