Abstract

This study describes the application of a composite autofocus method, which combines full-trip auto focusing (AF) technology with extended depth of field (EDOF) technology to examine tissue slides, using a line scan CCD camera. We investigated several criteria that serve as appropriate sharpness evaluation methods, for histological images. Examples of such methods include the use of an edge detection filter for the spatial domain and energy computation for the frequency domain. A tissue slice, taken for histological examination generally has a certain thickness, but due to the limitations in the depth of field (DOF) of the objective lens, it is not possible to ensure that the tissue section stays within the DOF of the objective lens, during the image acquisition process. Factors such as the thickness of the tissue slice, defects in the production process for the tissue slide and mechanical error impact the quality of the final slide. Our study focuses on the motion of the line scan CCD camera along the Z-axis, during the image acquisition process. Motion along the Z-axis allows for instant AF, during image acquisition. A guide curve, for Z/X-axis shift to the other layers, along with our proposals, can yield sharper images.

Key words: Autofocusing (AF), tissue slide, microscopy, histologic, line scan.