Microcomputer-based three-dimensional reconstruction of in situ hybridization autoradiographs

A number of software routines were written for the public domain Macintosh-based video-densitometry program, Image, to facilitate in situ hybridization analysis. These routines utilize fiducial marks drilled in unused portions of the brain to rotate high-magnification images of individual sections so that they may be placed in register. The regions of interest within the registered sections are outlined and intermediate sections interpolated to produce a data set representing a three-dimensional volume. This shell is then filled with the original density data obtained with one or more in situ hybridization probes. The final object can be viewed in various degrees of translucency and examined from different angles or computationally resectioned to yield new information concerning activity changes within the region and relationships between reactive sites. These static images can be collected and rapidly displayed as a movie. These routines have been used in this paper to display the differential hybridization of three different mRNA probes in the hypothalamic paraventricular nucleus.