Cho-Ying Chuang, Matthew Zammit, Miles L. Whitmore, Matthew J Comstock
We present an instrument that combines high-resolution optical tweezers and multi-color confocal fluorescence spectroscopy along with automated single molecule assembly. Multi-color allows the simultaneous observation of multiple molecules or multiple degrees of freedom which allows e.g., the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, with a reliable alignment scheme, that will allow three independent fluorescent probe or fluorescence resonance energy transfer (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 due to 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 5-fold. 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 multi-molecule complexes while preserving fluorophores.
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