Characterization of photodamage to Escherichia coli in optical traps
Characterization of photodamage to Escherichia coli in optical traps
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DOI：
10.1016/s0006-3495(99)77117-1
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发表时间：
1999-11-01
影响因子：
3.4
通讯作者：
Block, SM
中科院分区：
文献类型：
--
作者：
Neuman, KC;Chadd, EH;Block, SM
光镊（基于红外激光的光阱）已经成为分子和细胞生物学中的一种强大工具。然而，它们的有用性受到限制，特别是在体内，由捕获激光造成的标本损伤的可能性。关于这种现象的起源知之甚少，在这里，我们采用了一种波长可调的光学陷阱，其中显微镜物镜透射率在整个近红外范围内得到充分表征，并结合灵敏的旋转细菌细胞测定。通过单个鞭毛将大肠杆菌的单细胞系在盖玻片上：这些细胞以与其跨膜质子电位成比例的速率旋转（曼森等人，1980. J. Mol. 138：541-561）。监测受到激光照射的细胞的旋转速率允许对其代谢状态的快速和定量测量。采用该测定法，我们表征了整个有利于光捕获的近红外区域（790-1064 nm）的光损伤。光损伤的作用光谱在830和970 nm处表现出最小值，在870和930 nm处表现出最大值。在厌氧条件下，损伤降低到背景水平，这意味着氧参与了光损伤途径。光损伤的强度依赖性是线性的，支持单光子过程。这些发现可能有助于指导选择最适合光学捕获工作的激光器和实验方案。
Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon, Here we employed a wavelength tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (Manson et at., 1980. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm), The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.