Figure 1.

Optical layout and experimental scheme. A: Optical layout of the hybrid optical trap–TIRF‐instrument. Both the fluorescence excitation (532 nm, green) laser and the trapping laser (1,064 nm, red) were modulated by half wave plate (HWP) and polarizing beam splitters (PBS). A dichroic mirror (D1) was used to guide the two laser beams into the microscope from a side port. The scattered and non‐scattered light of the trapping laser from trapped beads is acquired at the quadrant photodiode (QPD) which reports the readout of applied forces. The desirable force is controlled by the displacement of biotin‐end ssNA tether to the trap center through an in‐house program adjusting the movement of nano‐stage. With objective‐type TIRF excitation, the fluorescence emission is back collected through objective and imaged onto an electro‐multiplying charge‐coupled device (EMCCD) camera. D1‐3, dichroic mirrors; F1‐F2, filters; T1, telescope. B: 5′‐end single‐stranded DNA is anchored onto polyethylene glycol (PEG) passivated coverglass surface through biotin‐neutravidin linkage and 3′‐tail of single‐stranded DNA is coupled to polystyrene bead by antigen–antibody interaction, digoxigenin (DIG), and anti‐digoxigenin (Anti‐DIG). Fluorescence excitation laser (green) excites the coverglass within a few hundred nanometers by TIR. During measurements, trapping laser (red) pulls out the linked bead and stretches single‐stranded nucleic acid linearly. NS3h is labeled with Cy3 dye as fluorescence tracking marker.