Single-molecule and ensemble FRET fluorescence measurements were performed with objective-type total internal reflection fluorescence (TIRF) microscopy on an inverted microscope (Nikon TiE) with an Apo TIRF 100× oil objective lens, numerical aperture 1.49 (Nikon) as described previously (14) and controlled using Micromanager (49). Samples were excited with 473-nm OBIS laser (Coherent), 532-nm (Crystalaser), or 635-nm (Blue Sky Research) lasers. For single-molecule data, emission for the FRET donor and emission channels were separated as previously described and recorded on an electron-multiplying charge-coupled device camera (Andor iXon) (15). For collection of the green fluorescent protein (GFP) signal, we used an additional set of emission filters mounted on a motorized flip mount (Thorlabs Inc.) placed the donor fluorescence emission path. Filters used included a 593/40 nm filter (Semrock Inc.) for the collection of donor emission, a 675/30 nm filter for the collection of acceptor emission, and a 514/30 nm filter (Semrock Inc.) for GFP emission collection. For ensemble FRET maps taken for whole cells, emitted light passed through a quad-edge laser-flat dichroic with center/bandwidths of 405/60 nm, 488/100 nm, 532/100 nm, and 635/100 nm from Semrock Inc. (Di01-R405/488/532/635-25×36) and corresponding quad-pass filter with center/bandwidths of 446/37 nm, 510/20 nm, 581/70 nm, 703/88 nm band-pass filter (FF01-446/510/581/703-25). GFP, donor, and acceptor images were taken through separate additional cubes stacked into the light path (GFP: 470/40 nm, 495 nm long-pass, and 525/50 nm; donor: 550 nm long-pass; acceptor: 679/41 nm and 700/75 nm) and recorded on a Hamamatsu Orca Flash 4.0 camera.

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