Wuite G J, Davenport R J, Rappaport A, Bustamante C
Department of Physics, University of California, Berkeley, California 94720 USA.
Biophys J. 2000 Aug;79(2):1155-67. doi: 10.1016/S0006-3495(00)76369-7.
We have developed an integrated laser trap/flow control video microscope for mechanical manipulation of single biopolymers. The instrument is automated to maximize experimental throughput. A single-beam optical trap capable of trapping micron-scale polystyrene beads in the middle of a 200-microm-deep microchamber is used, making it possible to insert a micropipette inside this chamber to hold a second bead by suction. Together, these beads function as easily exchangeable surfaces between which macromolecules of interest can be attached. A computer-controlled flow system is used to exchange the liquid in the chamber and to establish a flow rate with high precision. The flow and the optical trap can be used to exert forces on the beads, the displacements of which can be measured either by video microscopy or by laser deflection. To test the performance of this instrument, individual biotinylated DNA molecules were assembled between two streptavidin beads, and the DNA elasticity was characterized using both laser trap and flow forces. DNA extension under varying forces was measured by video microscopy. The combination of the flow system and video microscopy is a versatile design that is particularly useful for the study of systems susceptible to laser-induced damage. This capability was demonstrated by following the translocation of transcribing RNA polymerase up to 650 s.
我们开发了一种集成激光阱/流动控制视频显微镜，用于对单个生物聚合物进行机械操作。该仪器实现了自动化，以最大限度地提高实验通量。使用了一种单光束光阱，它能够在一个200微米深的微腔中间捕获微米级的聚苯乙烯珠子，这使得可以在该腔内插入一个微量移液器，通过吸力来固定第二个珠子。这些珠子共同作为易于交换的表面，感兴趣的大分子可以附着在它们之间。使用计算机控制的流动系统来交换腔内的液体并高精度地建立流速。流动和光阱可用于对珠子施加力，珠子的位移可以通过视频显微镜或激光偏转来测量。为了测试该仪器的性能，将单个生物素化的DNA分子组装在两个链霉亲和素珠子之间，并使用激光阱力和流体力来表征DNA的弹性。通过视频显微镜测量不同力作用下DNA的伸展情况。流动系统和视频显微镜的结合是一种通用的设计，对于研究易受激光诱导损伤的系统特别有用。通过跟踪转录RNA聚合酶长达650秒的转位，证明了这种能力。
