Single-molecule imaging was done live in GFP-PSEN1 or NCT-SNAP/GFP-PSEN1 in rescued PSEN1 sKO or PSEN1 and 2/NCT tKO MEFs, respectively, using Total Internal Reflection (TIRF) Microscopy on an Olympus IX71 microscope equipped with a 100 × 1.7 N.A. objective lens. Despite near-physiological levels of expression, levels were higher than the density limit of 2 spots/µm² for single-molecule imaging. Therefore, a circular region of 10–12 µm diameter in the center of the imaging field was photobleached using a focused laser steered by a set of scanning mirrors, similar to the PhotoGate approach (Madl et al., 2010 (link); Belyy et al., 2017 (link)). After the initial photobleaching, several rings were drawn with the focused laser at the edge of the bleached region at intervals of 5–10 s to control the re-population with unbleached molecules. For imaging, an iris in the TIRF illumination pathway was closed to eliminate glare from outside of the central region. Movies of 700 frames were recorded at 34 Hz with a back-illuminated EMCCD camera (Andor iXon DU-897).
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