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首页> 外文期刊>Journal of Microscopy >Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules.
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Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules.

机译：通过激光微束和光学镊子进行显微操作：从植物细胞到单个分子。

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Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.
机译：激光的完全操纵允许对植物细胞，亚细胞结构甚至单个DNA分子进行精确而温和的处理。近来，可负担得起的激光器已经可用于构造微束以及用于光镊。对于植物生物学家而言，这可能会引起人们对这些工具的新兴趣。在该杂志上回顾的早期实验表明，激光支持的材料向植物细胞或组织的显微注射可避免脆弱的玻璃毛细管在显微注射中遇到的机械问题。当在显微镜下观察时，植物原生质体可以彼此融合，并且产生三重或四重融合产物不是主要问题。在本文中，我们回顾了从根毛尖制备膜材料并在藻类中模拟微重力的实验。由于许多植物细胞是透明的，因此有可能在完整的活细胞内工作。新实验表明，可以通过具有空间分辨率的光学显微操作释放笼中化合物的细胞内钙，并研究钙的振荡。给出了禽心脏组织的示例，但该技术也适用于植物细胞研究。作为一种更具技术性的工具，可以使用光镊在空间上固定原本会在细胞内移动的亚细胞结构，从而即使在延长成像时间（例如在低荧光发射时）的情况下，也可以使用共聚焦显微镜对其进行研究。分子生物学的例子是通过激光微型工具处理染色体和分离的单个DNA分子。例如，可以沿着复杂的轨迹切割染色体，而不仅仅是垂直于它们的长轴。单个DNA分子被激光微束切割，然后将这种分子偶联到聚苯乙烯微珠上，然后以复杂的几何形状处理。在此，用常规荧光显微镜通过荧光染料如SYBRGreen使单个DNA分子可见。可以直接观察到限制性内切酶EcoRI的分子对单个DNA分子的切割，即，一种单分子限制性分析是可能的。最后，可以直接观察单个DNA分子的机械性能。

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