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High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy.

ABSTRACT: 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.

SUBMITTER: Whitley KD

PROVIDER: S-EPMC5540136 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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High-Resolution Optical Tweezers Combined With Single-Molecule Confocal Microscopy.

Whitley K D KD Comstock M J MJ Chemla Y R YR

Methods in enzymology 20161214

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 al ...[more]

PMID: 28062033

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