Femtosecond optoinjection of intact tobacco BY-2 cells using a reconfigurable photoporation platform.
Femtosecond optoinjection of intact tobacco BY-2 cells using a reconfigurable photoporation platform.
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DOI：
10.1371/journal.pone.0079235
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Dholakia K
中科院分区：
文献类型：
--
作者：
Mitchell CA;Kalies S;Cizmár T;Heisterkamp A;Torrance L;Roberts AG;Gunn-Moore FJ;Dholakia K
一个紧密聚焦的超短脉冲激光束入射到细胞膜上，先前已经显示出瞬时增加细胞膜的通透性，同时保持细胞的活力，一种被称为增透的技术。这种渗透性可用于帮助膜不可渗透的生物相关物质如染料、蛋白质和核酸进入细胞。超短脉冲激光已被证明是必不可少的，用于改造哺乳动物细胞，但它们很少被应用于植物细胞，由于其较大的尺寸和刚性和厚的细胞壁，这显着阻碍外源物质的细胞内输送。在这里，我们展示和量化飞秒光学注入膜不渗透染料到完整的BY-2烟草植物细胞培养生长，调查光学和生物参数。具体而言，我们表明，长轴向范围的传播不变（“无衍射”）贝塞尔光束，放松了对细胞膜上的紧密聚焦的要求，优于标准的高斯双折射光束，实现高达70%的光注入效率。对培养基渗透效应的研究表明，高渗细胞外培养基被发现是降低膨压和促进分子进入细胞所必需的。
A tightly-focused ultrashort pulsed laser beam incident upon a cell membrane has previously been shown to transiently increase cell membrane permeability while maintaining the viability of the cell, a technique known as photoporation. This permeability can be used to aid the passage of membrane-impermeable biologically-relevant substances such as dyes, proteins and nucleic acids into the cell. Ultrashort-pulsed lasers have proven to be indispensable for photoporating mammalian cells but they have rarely been applied to plant cells due to their larger sizes and rigid and thick cell walls, which significantly hinders the intracellular delivery of exogenous substances. Here we demonstrate and quantify femtosecond optical injection of membrane impermeable dyes into intact BY-2 tobacco plant cells growing in culture, investigating both optical and biological parameters. Specifically, we show that the long axial extent of a propagation invariant (“diffraction-free”) Bessel beam, which relaxes the requirements for tight focusing on the cell membrane, outperforms a standard Gaussian photoporation beam, achieving up to 70% optoinjection efficiency. Studies on the osmotic effects of culture media show that a hypertonic extracellular medium was found to be necessary to reduce turgor pressure and facilitate molecular entry into the cells.
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影响因子：
48
作者：
Barrett, Lindy E.;Sul, Jai-Yoon;Eberwine, James H.
通讯作者：
Eberwine, James H.
影响因子：
2.8
作者：
Antkowiak, Maciej;Torres-Mapa, Maria Leilani;Dholakia, Kishan
通讯作者：
Dholakia, Kishan
影响因子：
5.5
作者：
Ferrando, M;Spiess, WEL
通讯作者：
Spiess, WEL
影响因子：
38.3
作者：
通讯作者：
--
DOI：
10.1016/s0168-9002(02)00384-4
发表时间：
2002-05-01
影响因子：
1.4
作者：
Awazu, K;Kinpara, T;Tamiya, E
通讯作者：
Tamiya, E