Uplanapo 60 1.20 w objective

Observations of single-molecule fluorescence were made using a custom-built confocal microscope equipped with a continuous-wave 488 nm solid-state laser (FCD488–010, JDSU, Milpitas, CA, USA) and an Olympus UplanApo 60×/1.20W objective. After a dichroic mirror that separates excitation and emission light (500DCXR, Chroma Technology, Rockingham, VT, USA), fluorescence emission passed through a 100 𝜇m pinhole and was split by a second dichroic mirror (585DCXR, Chroma Technology) into donor and acceptor fluorescence. Donor fluorescence then passed a filter (ET525/50M, Chroma Technology) before being focused onto a single-photon avalanche diode (MPD 100ct, Micro Photon Devices, Bolzano, Italy) while acceptor fluorescence passed a filter (QT 650/100) before being focused onto a single-photon avalanche diode (SPCM-AQR-13, PerkinElmer Optoelectronics, Vaudreuil, QC, Canada). The arrival time of every photon was recorded with a two-channel time-correlated single-photon counting module (PicoHarp300, PicoQuant, Berlin, Germany). All measurements were performed with a laser power of 100 𝜇W, measured at the back aperture of the objective (beam waist: 8 mm).