Ldh d ta 530

After the preparation of immobilized, fluorophore-labeled SthK nanodiscs on the slide surface, sm-FRET data was acquired from individual SthK channels using a MicroTime 200 Fluorescence Lifetime Microscope from PicoQuant. A donor excitation laser (532 nm; LDH-D-TA-530; Picoquant, Berlin, Germany) and acceptor excitation laser (637 nm; LDH-D-C-640; Picoquant) were used with a Pulsed Interleaved Excitation (PIE) scheme to excite the fluorophores. Emitted photons were collected back through the objective lens (100×1.4 numerical aperture; Olympus). Emission filters for the donor (550 nm; FF01-582/64; AHF, Tübingen-Pfrondorf, Germany or Semrock, Rochester, NY) and acceptor (650 nm 2XH690/70; AHF) were used to select photons for each detection channel. Photons were then passed to two SPAD photodiodes (SPCM CD3516H, Excelitas technologies, Waltham, MA) to determine the fluorescence intensity for each fluorophore. The donor and acceptor fluorescence intensities over time were recorded for one SthK channel at a time and later analyzed as described below.

smFRET measurements were collected using a custom-built PicoQuant Microtime 200 Fluorescence Lifetime Microscope. To characterize the fluorescent behavior of both donor and acceptor fluorophores as well as the efficiency of the energy transfer between donors and acceptors, acquisition was conducted using both 532 nm (LDH-D-TA-530; Picoquant, Berlin, Germany) and 637 nm (LDH-D-C-640; Picoquant) lasers using pulsed interleaved excitation at 80 MHz. During scanning, the slide was immobilized on a scanning x-y-z piezo stage (P-733.2CD; Physik Instrumente) and observed through a 100× oil-immersed lens (100 × 1.4 NA; Olympus, Tokyo, Japan). Photons from samples post-excitation were then collected back through the objective and separated through a dual band dichroic beam splitter (Zt532/640rpc-UF3; AHF/Chroma, Bellows Falls, VT, USA). Prior to detection, photons were then filtered through emission filters (550 nm (FF01-582/64; AHF/Semrock, Rochester, NY, USA) for the donor or 650 nm (2XH690/70; AHF, Tübingen-Pfrondorf, Germany) for the acceptor) and into two SPAD photodiodes (SPCM CD3516H; Excelitas Technologies, Waltham, MA, USA).

Single molecule FRET measurements were acquired using a custom-built PicoQuant MicroTime 200 Fluorescence Lifetime Microscope. smFRET data acquisitions were conducted using pulsed interleaved excitation at 80 MHz. Both 532 nm (LDH-D-TA-530; Picoquant) and 637 nm (LDH-D-C-640; Picoquant) lasers were simultaneously used to characterize the fluorescent behavior of both fluorophores and the efficiency of energy transfer between molecules potentially showing FRET. The sample slide was immobilized on a scanning x-y-z piezo stage (P-733.2CD; Physik Instrumente) while being excited and observed through a 100x oil immersed lens (100 × 1.4 NA; Olympus). The photons emitted from the sample post-excitation were collected back through the objective, separated through a dual band dichroic beam splitter (Zt532/640rpc-UF3; AHF/Chroma) and sent to two SPAD photodiodes (SPCM CD3516H; Excelitas technologies) preceded by excitation filters. A 550 nm (FF01-582/64;AHF/Semrock) and 650 nm (2XH690/70;AHF) emission filter were used for the donor and acceptor channels, respectively. All acquisitions were performed in the presence of a photo-stabilizer and oxygen scavenging solution buffer system (ROXS).