Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores

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.