2.5. pTIRF Sample Preparation, Imaging, and Data Analysis

After mounting the functionalized flow cell on the stage of the prism-based total internal reflection fluorescence (pTIRF) microscope [56], a 200 µL solution of 20 pM biotinylated sensor construct prepared in an imaging buffer consisting of 10 mM MgCl₂ in 1× TAE and an oxygen scavenging system (4 mM Trolox, 10 mM PCA, 100 nM PCD) was injected. Through Mg²⁺ titration experiments, we determined that these sensors performed optimally at 10 mM Mg²⁺ (Figure S1). Therefore, all of the single molecule FRET experiments were performed at 10 mM MgCl₂. The flow cell was flushed with imaging buffer after 30 s of incubation in order to remove unbound sensor molecules. Data acquisition was performed at room temperature (23 °C) using Single.exe software, and post analysis was performed using IDL and MATLAB scripts, as described in our previous publications [31,53]. Using a 532 nm laser (power = ~32 mW), the Cy3 fluorophore was continuously excited throughout the movie (~200 s). Fluorescence emissions from Cy3 and Cy5 fluorophores were simultaneously recorded through green and red channels respectively (512 × 256 pixels) using an iXon Ultra EMCCD camera at a 100 ms time resolution. To confirm the presence of an active FRET pair in the sensor molecules, the red laser (λ = 639 nm, power = ~22 mW) was turned on after around 100 s of starting the movie. Those molecules showing evidence of both Cy3 and Cy5 with single-step photo-bleaching of the fluorophores were selected for further analysis. Finally, FRET histograms were plotted in Origin by binning the FRET data for the first 60 frames (all raw FRET data were combined from several molecules before binning) and fitted with one or a multi-peak Gaussian function. Although the movies were recorded for a long time window (~200 s), to include most of the molecules, we used the first 60 frames for binning the FRET data for histograms, as some molecules photobleach faster than others (Figure S2). The selected molecules were randomly assigned into three groups, and the three histograms obtained were used to estimate the mean FRET state, the FRET fractions (when relevant), and the standard deviations (SD). The number of replicates is identified in the figure legends, and the standard deviations are incorporated in the plots where applicable.