[show abstract][hide abstract] ABSTRACT: We describe a strategy for analyzing axonal transport of cytosolic proteins (CPs) using photoactivatable GFP-PAGFP-with modifications of standard imaging components that can be retroactively fitted to a conventional epifluorescence microscope. The photoactivation and visualization are nearly simultaneous, allowing studies of proteins with rapidly mobile fractions. Cultured hippocampal neurons are transfected with PAGFP-tagged constructs, a discrete protein population within axons is photoactivated, and then the activated population is tracked by live imaging. We show the utility of this method in analyzing axonal transport of CPs that have inherent diffusible pools and distinguish this transport modality from passive diffusion and vesicle transport. The analytical tools used to quantify the motion are also described. Aside from the time needed for preparation of neuronal cultures/transfection, the experiment takes 2-3 h, during which time several axons can be imaged and analyzed. These methods should be easy to adopt by most laboratories and may also be useful for monitoring CP movement in other cell types.