Optimal focusing with extended depth of focus in microscopic systems

Light microscopic systems have the limited depth of field. The objects into depth of focus only appear in-focus. When the samples observed at the microscope are thicker than depth of focus, it is seen that some regions on samples images are out of focus and a whole objects completely in one single image can't be obtained. In this study, it is aimed to prevent this problem and to produce an image with optimal focus in microscopic systems. In the literature, this process was achieved with extended depth of focus using images taken at random along the Z axis. In the proposed study, optimum focusing is provided with extended depth of focus, and the number of images in the image series used in this process is obtained a model. This study consists of three main stages: determination of reference image, selection of the image series and image fusion. The results obtained are qualitatively and quantitatively compared and in addition to studies in the literature, it is assured that the all regions of the samples are in depth of field. The proposed study was performed on the image sequences of the pleural effusion fluid prepared for cytopathological examination.