biostudies-literature00700Whitley KDNCRR NIH HHSNIGMS NIH HHS137-169https://www.ebi.ac.uk/biostudies/studies/S-EPMC5540136biostudies-literatureUnknown582We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument's capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme.Methods in enzymologyHigh-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy.PMC5540136R21 RR025341R01 GM120353Chemla YRComstock MJWhitley KD70falseHigh-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy.We describe the design, construction, and application of an instrument combining dual-trap, high-resolution optical tweezers and a confocal microscope. This hybrid instrument allows nanomechanical manipulation and measurement simultaneously with single-molecule fluorescence detection. We present the general design principles that overcome the challenges of maximizing optical trap resolution while maintaining single-molecule fluorescence sensitivity, and provide details on the construction and alignment of the instrument. This powerful new tool is just beginning to be applied to biological problems. We present step-by-step instructions on an application of this technique that highlights the instrument's capabilities, detecting conformational dynamics in a nucleic acid-processing enzyme.2017-01-01T00:00:00Z20172020-11-19T16:43:26Z2019-03-27T02:52:24ZS-EPMC55401362806203310.1016/bs.mie.2016.10.036