Uplsapo 60xo objective

Single-molecule FRET measurements were performed at room temperature using a home-built confocal microscope (as previously described 35 (link)). Briefly, the microscope operated with 20 kHz alternating-laser excitation between a 532-nm (Samba, Cobolt, operated at 240 μW) and a 638-nm laser (Cube, Coherent, operated at 60 μW), coupled to a 60×, 1.35 numerical aperture (NA), UPLSAPO 60XO objective (Olympus). Emitted photons were spectrally filtered and detected by two avalanche photo diodes (SPCM-AQRH-14, Perkin Elmer). The alternating laser excitation allows filtering for correctly labelled species bearing an active donor and an active acceptor (36 (link)). After filtering each fluorescent burst for the correct labelling stoichiometry, we calculated the apparent FRET efficiency E* for each burst as E* = DA/(DD + DA), where DA is the number of photons in the red detection channel after green excitation and DD the number of photons in green detection channel after green excitation.

Fig. 5A (which is the basis for Fig. 6A) and Fig. 6B were taken with a digital camera (Power Shot G12, Canon, Tokyo, Japan). The generation of Fig. 5C is described in detail above (BX51WI microscope; UPLSAPO20X objective; Olympus), as well as the generation of Fig. 6D and all panels in Figs 2, 3 and 7 (same microscope; UPLSAPO4X objective; Olympus). Fig. 5D was taken with the same microscope using an UPLSAPO60XO objective (60×, oil, N.A.=1.35) (Olympus). The final figures were constructed using Corel Photo-Paint X7 and Corel Draw X7 (both versions 17.5.0.907; Corel, Ottawa, Canada). Only minor adjustments of contrast and brightness were made using Corel Photo-Paint, without altering the appearance of the original materials.

Single-molecule FRET measurements were performed at room temperature using a home-built confocal microscope with 20 kHz alternating-laser excitation between a 532-nm (Samba, Cobolt, operated at 240 μW) and a 638-nm laser (Cube, Coherent, operated at 60 μW), coupled to a 60×, 1.35 numerical aperture (NA), UPLSAPO 60XO objective (Olympus) as previously described (11 (link)). For DNA–DNA measurements, labelled DNA was present at <100 pM and unlabelled Pol (when present) at 3 nM concentration. For Pol-DNA measurements, both Pol and DNA were present at 100 pM concentration. Measurements were taken in ‘Pol buffer’, consisting of 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–NaOH, pH 7.3, 10 mM MgCl₂, 1 mM DTT, 100 μg ml⁻¹ bovine serum albumin, 5% (v/v) glycerol, 1 mM mercaptoethylamine. Photon streams in DD, DA and AA channels were recorded and processed using custom-written software (LabVIEW). Bursts were filtered for the correct labelling stoichiometry (12 (link)), and accurate FRET efficiencies were calculated as described ((13 (link)) and Supplementary Methods). Distances were calculated from the FRET efficiencies, using experimentally determined Förster radii and donor quantum yields (Supplementary Table S4).