Dijk Meindert A van, Kapitein Lukas C, Mameren Joost van, Schmidt Christoph F, Peterman Erwin J G
Department of Physics, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.
J Phys Chem B. 2004 May 20;108(20):6479-84. doi: 10.1021/jp049805+.
To obtain high-resolution information on position or conformation of a molecule and at the same time apply forces to it, one can combine optical trapping with single-molecule fluorescence microscopy. The technical challenge in such an experiment is to discriminate a minute fluorescence signal from the much larger background signals caused by the trap and the fluorescence excitation laser light. We show here that this is feasible even when the fluorophore is directly attached to the trapped particle, by using optimized optical filters. We found, however, that the photostability of the fluorophores we tested suffered from the presence of the additional laser light used for trapping. We found that bleaching rates increased linearly with both the intensity of the trapping laser and the intensity of the fluorescence excitation light. Photobleaching rates were unaffected by the presence or absence of oxygen, but were significantly diminished in the presence of antioxidants. Our results indicate that the enhanced photobleaching is caused by the absorption of a visible photon followed by the excited-state absorption of a near-infrared photon. The higher excited singlet states generated in this way readily form nonfluorescent dye cations. We found that different dyes suffer to a different extent from the excited-state absorption, with Cy3 being worst and tetramethylrhodamine least affected.
为了获取分子位置或构象的高分辨率信息，同时对其施加力，可以将光镊与单分子荧光显微镜相结合。此类实验中的技术挑战在于，要从由光阱和荧光激发激光产生的大得多的背景信号中分辨出微弱的荧光信号。我们在此表明，通过使用优化的光学滤光片，即便荧光团直接附着在被捕获的粒子上，这也是可行的。然而，我们发现，我们测试的荧光团的光稳定性会受到用于捕获的额外激光的影响。我们发现，漂白速率随捕获激光强度和荧光激发光强度线性增加。光漂白速率不受氧气存在与否的影响，但在有抗氧化剂存在时会显著降低。我们的结果表明，增强的光漂白是由可见光子的吸收，随后是近红外光子的激发态吸收引起的。以这种方式产生的较高激发单重态很容易形成非荧光染料阳离子。我们发现，不同的染料受激发态吸收的影响程度不同，其中Cy3受影响最严重，而四甲基罗丹明受影响最小。
