Use of Software to Enhance Depth of Field and Improve Focus in Photomicrography

摘要

By using readily available free software or shareware, the focal depth and sharpness in photomicrographic images can be enhanced dramatically. In this way, digital images containing three-dimensional information are achievable, comparable with scanning electron micrographs. Moreover, several parameters which determine the quality of light microscope images, such as the illumination and imaging modes, can also be improved. To do this, a vertical stack of images is taken consisting of several single images with different planes of focus. The single images are then superimposed using the software; only regions that are in focus contribute to the resulting reconstructed image. The resulting images do not have any indistinctness, and are independent of the vertical dimension of the specimen and the magnification and focal depth of the objective.