Combined High-Resolution Optical Tweezers and Multicolor Single-Molecule Fluorescence with an Automated Single-Molecule Assembly Line.

We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single-molecule assembly. The multicolor allows the simultaneous observation of multiple molecules or multiple degrees of freedom, which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, with a reliable alignment scheme, which will allow three independent fluorescent probe or FRET measurements and also increases flexibility in the choice of fluorescent molecules. We demonstrate the ability to simultaneously measure angstrom-scale changes in tether extension and fluorescence signals. Simultaneous tweezers and fluorescence measurement are particularly challenging because of fluorophore photobleaching, even more so if multiple fluorophores are to be measured. Therefore, (1) fluorescence excitation and detection is interlaced with time-shared dual optical traps. (2) We investigated the photostability of common fluorophores. The mean number of photons emitted before bleaching was unaffected by the trap laser and decreased only slightly with increasing excitation laser intensity. Surprisingly, we found that Cy5 outperforms other commonly used fluorophores by more than fivefold. (3) We devised computer-controlled automation, which conserves fluorophore lifetime by quickly detecting fluorophore-labeled molecule binding, turning off lasers, and moving to add the next fluorophore-labeled component. The single-molecule assembly line enables the precise assembly of multimolecule complexes while preserving fluorophores.