I pentamax

iSiMREPS experiments were performed using either an Olympus IX-71 prism-type TIRF microscope equipped with a 60× water-immersion objective (Olympus UPLANAPO, 1.2NA) or an Olympus IX-81 objective-type TIRF microscope equipped with a 60× oil-immersion objective (APON 60XOTIRF, 1.49 NA) with CellTIRF and z-drift control modules. An ICCD (I-Pentamax, Princeton Instruments, MCP Gain 60) or sCMOS (Hamamatsu C13440-20CU) camera was used to record movies for the prism-TIRF while an EMCCD camera (IXon 897, Andor, EM gain 150) was used for the objective-TIRF. For recording smFRET signal, the Cy3-Alexa Fluor 647 fluorophore pairs were excited by light from a 532 nm laser at a power of 15–30 mW. For reliably detecting FRET signals with satisfactory S/N, an illumination intensity of ~50 W/cm² is typically used, and the TIRF angle adjusted to achieve a calculated evanescent field penetration depth of ~70–85 nm. Two-channel images were recorded using a prism-TIRF microscope while only acceptor channel images were recorded using an objective-TIRF microscope. In prism-TIRF imaging, the signal integration time (exposure time) per frame was 100 ms, laser power was ~ 18 mW, and movies ranging from 1–15 minutes were collected to assess FRET behavior comprehensively. In objective-TIRF imaging, the exposure time per frame was 60–100 ms, and typically 200–600 movie frames were acquired per FOV.