The principle and applications of optical microscope tomography.

A new principle to obtain sectional images of a thick specimen by a conventional optical microscope without physically slicing is developed. An optical microscope with an off-axis pupil is used to project a three-dimensional (3-D) distribution of a specimen in various directions within the numerical aperture of the objective lens onto a fixed TV camera position. The obtained images of the thick sample are combined to reconstruct its 3-D density distribution. Since the system is strictly angularly-limited, a strong constraint is needed to form the 3-D structure with a moderate spatial resolution from the projections. The conjugate gradient method with the object boundary constraint which is a priori known solves this problem. Experimental results with a biological sample verify the capability of the proposed method as a promising instrumentation of optical tomography in microscopic size.