FLIM demonstrates that tagged SPP constructs form dimers in intact cells. a). Confocal microscopy images of Alexa 488 labeled SPPCTV5 and Cy3 labeled SPPNTFLAG in stably transfected HEK cells demonstrate the cellular distribution of SPP. b). FLIM was then used to monitor the proximity of Alexa 488 labeled SPPCTV5 and Cy3 labeled SPPNTFLAG molecules. Intensity images (A,C) again show the immunostaining of Alexa 488 labeled SPPCTV5 (donor fluorophore). The corresponding FLIM images provide the visualization of the degree of donor fluorophore lifetime shortening on a pseudocolor scale (B,D). B) Alexa 488 lifetime distribution in the absence of an acceptor fluorophore (negative control). D) The red pixels represent shortening of the donor fluorophore lifetime indicating that two SPP molecules form a dimer and come into closest proximity at the periphery of the cell. c) Confocal microscopy images of cells stably expressing SPPCTV5 were labeled by goat anti-V5 Alexa 488 and Cy3 anti-goat IgG and demonstrate that the two fluorophores are in close proximity of one another (positive control). A) The intensity image of SPPCTV5 Alexa 488 immunoreactivity. B) The pseudocolored FLIM image shows shortened Alexa 488 lifetime (~1300 psec, red pixels) ubiquitously distributed throughout the cell.