Simultaneous Multiplane Imaging in Digital Holographic Microscopy

Reduced depth of field is a severe limitation in microscopy since, at high magnification, only a portion of the imaged volume along the optical axis is in good focus at once. We demonstrate that, adopting a numerical quadratic deformed diffraction grating into the reconstruction process of digital holograms, it is possible to image three planes at different depths simultaneously, gaining flexibility dispensing with a physical grating. Moreover, multiplexed holograms recorded with two different wavelengths can be obtained in focus simultaneously by a single reconstruction. Description of the method and applications for macroscopic and microscopic biological samples are reported.