An automatic method that fully compensates the quantitative phase measurements in off-axis digital holographic microscopy (DHM) is presented. The two main perturbations of the quantitative phase measurements in off-axis DHM are automatically removed. While the curvature phase flaw introduced by the microscope objective is avoided by the use of an optimized telecentric imaging system for the recording of the holograms, the remaining phase perturbation due to the tilt of the reference wave is removed by the automatic computation of a digital compensating reference wave. The method has been tested on both nonbiological and biological samples with and improving on the quality of the recovered phase maps.

The description and validation of an ImageJ open-source plugin to numerically simulate and reconstruct digital lensless holographic microscopy (DLHM) holograms are presented. Two modules compose the presented plugin: the simulation module implements a discrete version of the Rayleigh–Somerfield diffraction formula, which allows the user to directly build a simulated hologram from a known phase and/or amplitude object by just introducing the geometry parameters of the simulated setup; the plugin’s reconstruction module implements a discrete version of the Kirchhoff–Helmholtz diffraction integral, thus allowing the user to reconstruct DLHM holograms by introducing the parameters of the acquisition setup and the desired reconstruction distance. The plugin offers the two said modules within the robust environment provided by a complete set of built-in tools for image processing available in ImageJ. While the simulation module has been validated through the evaluation of the forecasted lateral resolution of a DLHM setup in terms of the numerical aperture, the reconstruction module is tested by means of reconstructing experimental DLHM holograms of biological samples.

A self-focusing method applicable to digital lensless holographic microscopy is presented. The method searches for the global minimum of the area enclosing a given amount of energy in a region surrounding the object of interest. The proposed modified enclosed energy method has been tested on self-focusing experimental holograms of a paramecium specimen and a section of the head of a drosophila melanogaster fly. The presented self-focusing technique also has been contrasted with some of the already reported methods to seek the best focus image.

pyDHM is an open-source Python library aimed at Digital Holographic Microscopy (DHM) applications. The pyDHM is a user-friendly library written in the robust programming language of Python that provides a set of numerical processing algorithms for reconstructing amplitude and phase images for a broad range of optical DHM configurations. The pyDHM implements phase-shifting approaches for in-line and slightly off-axis systems and enables phase compensation for telecentric and non-telecentric systems. In addition, pyDHM includes three propagation algorithms for numerical focusing complex amplitude distributions in DHM and digital holography (DH) setups. We have validated the library using numerical and experimental holograms.

A holographic point source (HPS) developed for digital lensless holographic microscopy (HPS-DLHM) is presented. The HPS is an off-axis phase transmission hologram of an experimental micrometer pinhole recorded on a photopolymer holographic film. An amplitude division interferometer, adjusted to operate at maximum diffraction efficiency, has been employed to record the hologram. The results of HPS-DLHM have been contrasted with the results obtained via conventional DLHM, and the two techniques were found to give similar measurements. Compared with conventional pinhole-based DLHM illumination, our cost-effective proposal provides increased mechanical stability, the possibility of wider spherical illumination cones, and shorter reconstruction distances. These superior features pave the way to applying this quantitative phase imaging (QPI) technique in biomedical and telemedicine applications. The imaging capabilities of our HPS-DLHM proposal have been tested by using an intricate sample of a honeybee leg, a low-absorption sample of epithelial cheek cells, a 1951 USAF test target, and smeared human erythrocytes.