Orthogonal‐plane fluorescence optical sectioning: Three‐dimensional imaging of macroscopic biological specimens

An imaging technique called orthogonal-plane fluorescence optical sectioning (OPFOS) was developed to image the internal architecture of the cochlea. Expressions for the three-dimensional point spread function and the axial and lateral resolution are derived. Methodologies for tissue preparation and for construction, alignment, calibration and characterization of an OPFOS apparatus are presented. The instrument described produced focused, high-resolution images of optical sections of an intact, excised guinea-pig cochlea. The lateral and axial resolutions of the images were 10 and 26 microns, respectively, within a 1.5-mm field of view.