Fig. 5. Imaging of Tf and EEA1. (A) COS-7 cells expressing GFP-EEA1 (green) were exposed to 10 µg/ml Alexa-Fluor-568-Tf (red) for the times indicated in each panel. After 60 seconds cells were washed, incubated with 200 µg/ml unlabeled Tf, and imaged by TIRF-M with an incident angle set to visualize 300 nm from the coverslip. Colocalized voxels are depicted in white. (B) Higher resolution series depicting the transient nature of the colocalization of Tf with EEA1. (C) TIRF images were obtained at 0.5 Hz, and at 60-second intervals illumination was switched to epifluorescence for the acquisition of optical sections at 250 nm intervals through the entire cell volume. Shown is the TIRF image preceding the acquisition of the image stack. Optical sections were projected into a single 2D image. Arrows in A and B indicate the movement of Tf-containing vesicles as they near EEA1-enriched endosomes; however, colocalization is brief because the Tf structures move away from the EEA1 signal.