Digital holographic microscopy and focusing methods based on image sharpness.

Digital holographic microscope allows imaging of opaque and transparent specimens without staining. A digitally recorded hologram must be reconstructed numerically at the actual depth of the object to obtain a focused image. We have developed a high-resolution digital holographic microscope for imaging amplitude and phase objects with autofocusing capability. If the actual depth of an object is not known a priori, it is estimated by comparing the sharpness of several reconstructions at different distances, which is very demanding in means of computational power when the recorded hologram is large. In this paper, we present 11 different sharpness metrics for estimating the actual focus depths of objects. The speed performance of focusing is discussed, and a scaling technique is introduced where the speed of autofocusing increases on the order of square of the scale ratio. We measured the performance of scaling on computer-generated holograms and on recorded holograms of a biological sample. We show that simulations are in good agreement with the experimental results.