Confocal microscopy, a key tool in neuroscience, is designed to provide 3D information. However, in practice, analysis of multiple labeling is almost exclusively performed on 2D images, reflecting the lack of effective tools for analyzing multi-channel images in 3D. For this reason we are developing a new software application, ImageSurfer, an initial version of which was reported at the 2004 SFN meeting. ImageSurfer is an interactive software package for volume visualization and data analysis that enables researchers to quickly explore complex 3D confocal images. It is designed to be user-friendly and intuitive with a gentle learning curve. ImageSurfer incorporates traditional visualization tools such as maximum intensity projection, direct volume rendering, and isosurface rendering; and advanced tools to analyze and quantify relationships between multi-channel confocal images. A specialized colored isosurface technique permits display of an easily-understood 3D view showing the correspondence between two ...