Depth-of-field extension and 3D reconstruction in digital holographic microscopy

The limited depth-of-field is a main drawback of microscopy that prevents from observing, for example, thick semi-transparent objects with all their features in focus. Several algorithms have been developed during the past years to fuse images having various planes of focus and thus obtain a completely focused image with virtually extended depth-of-field. We present a comparison of several of these methods in the particular field of digital holographic microscopy, taking advantage of some of the main properties of holography. We especially study the extended depth-of-field for phase images reconstructed from the hologram of a biological specimen. A criterion of spatial measurement on the object is considered, completed with a visual criterion. The step of distance taken into account to build the stack of images is less than the instrument depth-of-field. Then, preserving the distance of focus associated with each pixel of the image, a three-dimensional representation is presented after automatic detection of the object. The limits of such a method of extraction of 3D information are discussed.