Three-dimensional imaging through millimeter-thick tissue specimens

A 780 nm stage-scanned confocal transmission microscope using compact disk (CD) player optics has been constructed and used to study test objects (including scattering effects) and breast tissue specimens (25-1500 micrometer thick). Confocal alignment in transmission is achieved using the CD optical mechanism moving coils to scan the objective lens until intensity peaks; the specimen is present but motionless. Intensity variation as a 3D function of mis- alignment of the two foci may allow estimation of optic deterioration caused by the specimen; appropriate confocal apertures and sample spacings can then be selected. Experimental measurements have been supplemented by 2D Monte-Carlo modeling of photon transport for a scattering- limited confocal transmission system. Modeling has characterized: transmission as a function of focal depth in a slab; contrast produced by a range of embedded spherical target radii; and optical mis-alignment. Poor dye perfusion within thick stained specimens results in little visible internal structure. This may be improved using partially phase dependent imaging (approximately equal to split- detector), sensitive to specimen refractive index variations.