In polarized mature neurons, the asymmetrical distribution of proteins between axonal and somatodendritic plasma membrane (PM) domains may be maintained by a diffusion barrier at the axon-soma boundary. At the boundary, a complex containing membrane-associated and cytoskeletal proteins is formed, anchoring axonal membrane proteins and indirectly hindering the diffusion of other membrane proteins. We examined the latter case, i.e., secondary diffusion impedance by comparing the mobility of fluorescently labeled membrane proteins within the axon-soma and dendrite-soma boundaries. We performed fluorescence recovery after photobleaching (FRAP) experiments using mature cultured hippocampal neurons that had been labeled specifically at their PMs with fluorescent proteins (FPs). The maturation of these neurons was confirmed by immunolocalization with Ankyrin-G, which is thought to participate in the creation of the diffusion barrier at the axonsoma boundary. We developed a wide-field microscope equipped with a device (digital micromirror device) composed of 1024 x 768 binary mirrors at the field-stop, allowing free control of the illumination area and intensity. After the FPs in peripheral processes were photobleached, nonbleached FPs diffused into all the processes at equivalent speeds. These results indicate that the secondary diffusion barrier to exogenously overexpressed membrane proteins is not specific to the axon-soma boundary. (C) 2003 Elsevier Inc. All rights reserved.