ImageSurfer is a free 3D imaging program for microcopy. It allows investigators to visualize and analyze multi-channel 3D and 4D (time-lapse) volumes. ImageSurfer provides several image restoration tools including deconvolution algorithms (Wiener filter, constrained iterative and maximum likelihood) to remove blurring introduced by the imaging system. For visualization, ImageSurfer provides several volume and surface rendering techniques. The software also has an innovative colored isosurface mode that provides a qualitative display of the correspondence between two channels. A "slice extractor" enables the user to examine a cross section of the volume taken at any orientation. The captured slice can be visualized as a traditional 2D image or as a 3D-relief representation in which the image intensity is coded as height. To further probe spatial overlap between structures, the investigator can use data from another channel to colorize the 3D relief representation. For quantification, signal intensity values can then be sampled within captured slices along a user-defined spline. In addition to taking snapshots of 3D representations and generating movies, 3D models can also be exported as OBJ or VRML (virtual reality modeling language) files; VRML files can be viewed within Web browsers using a free plug-in. Both file types can be imported into popular 3D rendering programs for further processing, and into Acrobat 3D to produce documents that can be viewed interactively with Adobe Reader 8.