Ultrahigh-resolution optical trap with single-fluorophore sensitivity

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore-labeled DNDNA oligonucleotides to bind and unbind complementary DNDNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNDNA-binding proteins (for example, single-base-pair stepping of DNDNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).