Automated high-resolution three-dimensional fluorescence imaging of large biological specimens.

We describe a novel automated technique for visualizing the three-dimensional distribution of fluorochrome-labelled components, in which image resolution is uncoupled from specimen size. This method is based on computer numerically controlled milling technology and combines an arrayed imaging technique with fluorescence capabilities. Fluorescent signals are segmented by emission spectra such that multiple fluorochromes present within a single specimen may be reconstructed and visualized individually or as a group. The automated nature of the system minimizes the workload and time involved in image capture and volume reconstruction. As an application, the system was used to image zones of fluorochrome-labelled microdamage within an 8-mm diameter cylinder of trabecular bone at a voxel size of 3 x 3 x 8 microm3. Our reconstruction of this specimen provides a visual map and quantitative measures of the volume of damage present throughout the cylinder, clearly demonstrating the interpretive power afforded by three-dimensional visualization. The three-dimensional nature of this highly automated and adaptable system has the potential to facilitate new diagnostic tools and techniques with application to a wide range of biological and medical research fields.