Stimulation of living cells by femtosecond near-infrared laser pulses
Stimulation of living cells by femtosecond near-infrared laser pulses
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DOI：
10.1117/12.478591
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发表时间：
2003-06
期刊：
影响因子：
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通讯作者：
S. Iwanaga;N. Smith;K. Fujita;T. Kaneko;M. Oyamada;T. Takamatsu;S. Kawata;O. Nakamura
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文献类型：
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作者：
S. Iwanaga;N. Smith;K. Fujita;T. Kaneko;M. Oyamada;T. Takamatsu;S. Kawata;O. Nakamura
我们已经证明，飞秒近红外激光脉冲可以诱导活 HeLa 细胞中的细胞内 Ca2+ 波。在本文中，我们介绍了使用药理学方法对 Ca2+ 波产生过程进行研究的结果，以确定产生机制。使用锁模钛宝石激光器（780 nm，80 fs，82 MHz）作为波触发光源。使用水浸物镜（NA 0.9）将激光束聚焦到 HeLa 细胞中。使用荧光 Ca2+ 指示剂 (Fluo-4) 观察 Ca2+ 波，并通过荧光显微镜进行监测。考虑了 Ca2+ 波产生的三种机制； (1) Ca2+ 通过破坏细胞膜流入细胞，(2) 与激光吸收相关的冲击波产生机械应力，(3) 通过破坏细胞内 Ca2+ 储备而泄漏 Ca2+。为了研究其机制，我们进行了实验以确定Ca2+波产生概率与两种细胞外溶液的依赖性； (a) 不含 Ca2+ 的细胞外溶液（通过使用 EGTA），以及 (b) 含有 U-73122 的溶液，可抑制对基于冲击波的机械效应的响应。从这些实验结果，我们可以得出结论，激光照射产生Ca2+波的主要机制是由于细胞内Ca2+储存被破坏而导致Ca2+泄漏。
We have demonstrated that intracellular Ca2+ waves in a living HeLa cell can be induced by femtosecond near-infrared laser pulses. In this paper, we present the results of investigation on the process of the Ca2+ wave generation using pharmacological methods to determine generation mechanisms. A mode-locked Ti:Sapphire laser (780 nm, 80 fs, 82 MHz) was used as a wave-triggering light source. The laser beam was focused into HeLa cells by using a water immersion objective lens (NA 0.9). Ca2+ waves were visualized by using a fluorescent Ca2+ indicator (Fluo-4) and monitored by a fluorescence microscope. Three mechanisms for the Ca2+ wave generation were considered; (1) Ca2+ flow into cells by destruction of the cell membrane, (2) mechanical stress by shock waves associated with the laser absorption, and (3) the leaking of Ca2+ through the destruction of intracellular Ca2+ stores. To investigate the mechanisms, we have performed experiments to determine the dependence of the probability of Ca2+ wave generation with two kinds of extracellular solutions; (a) a Ca2+ free extracellular solution (by use of EGTA), and (b) a solution containing U-73122 to inhibit the response to shockwave-based mechanical effects. From these experimental results, we can conclude the main mechanism of Ca2+ wave generation by laser irradiation is due to the leaking of Ca2+ through the destruction of intracellular Ca2+ stores.