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Digital holographic microscopy (DHM) is a cutting-edge interferometric technique to recover the complex wavefield scattered by microscopic samples from digitally recorded intensity patterns. In off-axis DHM, the challenge is digitally generating the reference wavefront replica to compensate for the tilt between the interfering waves. Current methods to estimate the reference wavefront's parameters rely on brute-force grid searches or heuristics algorithms. Whereas brute-forced searches are time-consuming and impractical for video-rate quantitative phase imaging and analysis, applying heuristics methods in holographic videos is limited since the phase background level occasionally changes between frames. A semi-heuristic phase compensation (SHPC) algorithm is proposed to address these challenges to reconstruct phase images with minimum distortion in the full field-of-view (FOV) from holograms recorded by a telecentric off-axis digital holographic microscope. The method is tested with a USAF test target, smearing red blood cells and alive human sperm. The SHPC method provides accurate phase maps as the reference brute-force method but with a 92-fold reduction in processing time. Furthermore, this method was tested for reconstructing experimental holographic videos of dynamic specimens, obtaining stable phase values and minimal differences in the background between frames. This proposed method provides state-of-the-art phase reconstructions with high accuracy and stability in holographic videos, allowing the successful XYZ tracking of single-moving sperm cells. 

Digital holographic microscopy (DHM) enables the three-dimensional (3D) reconstruction of quantitative phase distributions from a defocused hologram. Traditional computational algorithms follow a sequential approach in which one first reconstructs the complex amplitude distribution and later applies focusing algorithms to provide an in-focus phase map. In this work, we have developed a synergistic computational framework to compensate for the linear tilt introduced in off-axis DHM systems and autofocus the defocused holograms by minimizing a cost function, providing in-focus reconstructed phase images without phase distortions. The proposed computational tool has been validated in defocused holograms of human red blood cells and three-dimensional images of dynamic sperm cells. 

We present a fast algorithm for accurate phase compensation of holographic videos of dynamic phenomena recorded in a digital holographic microscope. The proposal is tested with human sperm and red blood cell samples. 

Digital holographic microscopes operating in non-telecentric mode introduce a spherical wavefront that distorts the sample's phase map, requiring its compensation for undistorted phase measurements. We have developed an open-access reconstruction tool that reconstructs non-telecentric holograms 

This outreach undergraduate research project presents a low-cost method to distinguish the quality of different olive oils. The proposed method is based on an indirect measurement of the chlorophyll molecules present when a green laser diode illuminates the oil sample. Oil blends can be classified into five classes (no olive oil, light olive oil, medium olive oil, olive oil, and extra virgin olive oil) by quantifying the ratio of the red channel versus the green channel along the laser illumination path from a color image. After labeling each oil blend, a convolutional neural network has been implemented and trained to automatically classify oil blends from a color image. The trained convolutional neural network has an accuracy of 90% in identifying and categorizing oil blends. This undergraduate research project introduces students to an interdisciplinary application requiring the combination of optical spectroscopy (i.e., multicolor imaging), image processing, and machine learning. In addition, due to the simplicity of the optical apparatus and computational analysis, high school students could implement and validate their own costeffective oil-quality classification device. 

Quantitative phase imaging (QPI) via Digital Holographic microscopy (DHM) has been widely applied in material and biological applications. The performance of DHM technologies relies heavily on computational reconstruction methods to provide accurate phase measurements. Among the optical configuration of the imaging system in DHM, imaging systems operating in a non-telecentric regime are the most common ones. Nonetheless, the spherical wavefront introduced by the non-telecentric DHM system must be compensated to provide undistorted phase measurements. The proposed reconstruction approach is based on previous work from Kemper’s group. Here, we have reformulated the problem, reducing the number of required parameters needed for reconstructing phase images to the sensor pixel size and source wavelength. The developed computational algorithm can be divided into six main steps. In the first step, the selection of the +1-diffraction order in the hologram spectrum. The interference angle is obtained from the selected +1 order. Secondly, the curvature of the spherical wavefront distorting the sample’s phase map is estimated by analyzing the size of the selected +1 order in the hologram’s spectrum. The third and fourth steps are the spatial filtering of the +1 order and the compensation of the interference angle. The next step involves the estimation of the center of the spherical wavefront. An optional final optimization step has been included to fine-tune the estimated parameters and provide fully compensated phase images. Because the proper implementation of a framework is critical to achieve successful results, we have explicitly described the steps, including functions and toolboxes, required for reconstructing phase images without distortions. As a result, we have provided open-access codes and a user interface tool with minimum user input to reconstruct holograms recorded in a non-telecentric DHM system. 

Confocal microscopes are known for optically removing the out-of-focus information (i.e., blur) in each transverse section of the sample’s volume, providing a more accurate three-dimensional image of thick microscopic samples than widefield microscopes. In this work, we have designed and evaluated a confocal microscope using off-of-shelf optical components from Thorlabs’ catalog, one of the major optical manufacturers. The design and evaluation have been implemented using Zemax OpticStudio, the standard optical system design software for realistic optical simulation. Here, we have also reported a practical protocol for building a confocal microscope using sequential mode. 

We have developed a joint phase compensation and autofocusing method for telecentric off-axis Digital Holographic Microscopy (DHM), providing in-focus reconstructed phase images without phase distortions. 

This research undergraduate study evaluates the accuracy and robustness of a PCA-based phase reconstruction algorithm based on the number of phase-shifted images and the phase step for an in-line digital holographic microscope. 

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.