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.
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Video-Rate Quantitative Phase Imaging Using a Digital Holographic Microscope and a Generative Adversarial Network
The conventional reconstruction method of off-axis digital holographic microscopy (DHM) relies on computational processing that involves spatial filtering of the sample spectrum and tilt compensation between the interfering waves to accurately reconstruct the phase of a biological sample. Additional computational procedures such as numerical focusing may be needed to reconstruct free-of-distortion quantitative phase images based on the optical configuration of the DHM system. Regardless of the implementation, any DHM computational processing leads to long processing times, hampering the use of DHM for video-rate renderings of dynamic biological processes. In this study, we report on a conditional generative adversarial network (cGAN) for robust and fast quantitative phase imaging in DHM. The reconstructed phase images provided by the GAN model present stable background levels, enhancing the visualization of the specimens for different experimental conditions in which the conventional approach often fails. The proposed learning-based method was trained and validated using human red blood cells recorded on an off-axis Mach–Zehnder DHM system. After proper training, the proposed GAN yields a computationally efficient method, reconstructing DHM images seven times faster than conventional computational approaches.
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- Award ID(s):
- 2042563
- NSF-PAR ID:
- 10315787
- Date Published:
- Journal Name:
- Sensors
- Volume:
- 21
- Issue:
- 23
- ISSN:
- 1424-8220
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Grulkowski, Ireneusz (Ed.)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.more » « less
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This works presents a reconstruction algorithm to recover the complex object information for an off-axis digital holographic microscope (DHM) operating in the telecentric regimen. We introduce an automatic and fast method to minimize a cost function that finds the best numerical conjugated reference beam to compensate the filtered object information, eliminating any undesired phase perturbation due to the tilt between the reference and object waves. The novelties of the proposed approach, to the best of our knowledge, are a precise estimation of the interference angle between the object and reference waves, reconstructed phase images without phase perturbations, and reduced processing time. The method has been validated using a manufactured phase target and biological samples.
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Off-axis digital holographic microscopy (DHM) provides both amplitude and phase images, and so it may be used for label-free 3D tracking of micro- and nano-sized particles of different compositions, including biological cells, strongly absorbing particles, and strongly scattering particles. Contrast is provided by differences in either the real or imaginary parts of the refractive index (phase contrast and absorption) and/or by scattering. While numerous studies have focused on phase contrast and improving resolution in DHM, particularly axial resolution, absent have been studies quantifying the limits of detection for unresolved particles. This limit has important implications for microbial detection, including in life-detection missions for space flight. Here we examine the limits of detection of nanosized particles as a function of particle optical properties, microscope optics (including camera well depth and substrate), and data processing techniques and find that DHM provides contrast in both amplitude and phase for unresolved spheres, in rough agreement with Mie theory scattering cross-sections. Amplitude reconstructions are more useful than phase for low-index spheres and should not be neglected in DHM analysis.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/s21238021
