Fluorescence Lifetime Imaging Microscopy (FLIM)

The relative proximity between fluorescently labeled N-terminus and the cytoplasmic loop domain between the transmembrane helices 6 and 7 of the endogenous PS1 was determined by previously validated FLIM assay for PS1 conformation (11). Briefly, pulsing Chameleon Ti:Sapphire laser (Coherent Inc.) was used to excite Alexa Fluor 488 donor fluorophore (two-photon excitation at 780-nm wavelength). The lifetimes of the donor fluorophore were recorded using a high-speed photomultiplier tube (MCP R3809; Hamamatsu) and a fast time-correlated single-photon counting acquisition board (SPC-830; Becker & Hickl). The baseline lifetime of the donor fluorophore (Alexa Fluor 488) in the absence of the acceptor (Cy3) was determined as the Förster resonance energy transfer (FRET) negative control (τ1). The presence of acceptor fluorophore < 10 nm from the donor results in FRET and a characteristic shortening of the donor fluorophore lifetime (τ2). The degree of the donor lifetime shortening correlates with the relative proximity between the fluorophores. The percent FRET efficiency (E_FRET%) is calculated as follow: E_FRET% = (τ1-τ2)/τ1*100%. The images were acquired using Zeiss LSM510 confocal microscope with ZEN 2009 software equipped with Zeiss 63x/1.4 Oil DIC objective. The data were analyzed using SPC Image software (Becker & Hickl).