Advanced Quantitative Phase Microscopy Achieved with Spatial Multiplexing and a Metasurface.

Quantitative differential phase-contrast (DPC) imaging is one of the commonly used methods for phase retrieval. However, quantitative DPC imaging requires several pairwise intensity measurements, which makes it difficult to monitor living cells in real-time. In this study, we present a single-shot quantitative DPC imaging method based on the combination of deep learning (DL) and color-encoded illumination. Our goal is to train a model that can generate an isotropic quantitative phase image (i.e., target) directly from a single-shot intensity measurement (i.e., input). The target phase image was reconstructed using a linear-gradient pupil with two-axis measurements, and the model input was the measured color intensities obtained from a radially asymmetric color-encoded illumination pattern. The DL-based model was trained, validated, and tested using thirteen different cell lines. The total number of training, validation, and testing images was 264 (10 cells), 10 (1 cell), and 40 (2 cells), respectively. Our results show that the DL-based phase images are visually similar to the ground-truth phase images and have a high structural similarity index (>0.98). Moreover, the phase difference between the ground-truth and DL-based phase images was smaller than 13%. Our study shows the feasibility of using DL to generate quantitative phase imaging from a single-shot intensity measurement.

Photoacoustic (PA) imaging systems are spreading in the biomedical community, and the development of new PA contrast agents is an active area of research. However, PA contrast agents are usually characterized with spectrophotometry or uncalibrated PA imaging systems, leading to partial assessment of their PA efficiency. To enable quantitative PA spectroscopy of contrast agents in vitro with conventional PA imaging systems, we have developed an adapted calibration method. Contrast agents in solution are injected in a dedicated non-scattering tube phantom imaged at different optical wavelengths. The calibration method uses a reference solution of cupric sulfate to simultaneously correct for the spectral energy distribution of excitation light at the tube location and perform a conversion of the tube amplitude in the image from arbitrary to spectroscopic units. The method does not require any precise alignment and provides quantitative PA spectra, even with non-uniform illumination and ultrasound sensitivity. It was implemented on a conventional imaging setup based on a tunable laser operating between 680 nm and 980 nm and a 5 MHz clinical ultrasound array. We demonstrated robust calibrated PA spectroscopy with sample volumes as low as 15 μL of known chromophores and commonly used contrast agents. The validated method will be an essential and accessible tool for the development of new and efficient PA contrast agents by improving their quantitative characterization.

We present single-shot high-performance quantitative phase imaging with a physics-inspired plug-and-play denoiser for polarization differential interference contrast (PDIC) microscopy. The quantitative phase is recovered by the alternating direction method of multipliers (ADMM), balancing total variance regularization and a pre-trained dense residual U-net (DRUNet) denoiser. The custom DRUNet uses the Tanh activation function to guarantee the symmetry requirement for phase retrieval. In addition, we introduce an adaptive strategy accelerating convergence and explicitly incorporating measurement noise. After validating this deep denoiser-enhanced PDIC microscopy on simulated data and phantom experiments, we demonstrated high-performance phase imaging of histological tissue sections. The phase retrieval by the denoiser-enhanced PDIC microscopy achieves significantly higher quality and accuracy than the solution based on Fourier transforms or the iterative solution with total variance regularization alone.

Microscopy combined with the transport of intensity equation is capable of retrieving both intensity and phase distributions of samples from both in-focus and defocus intensities. However, during measurements, the focal plane is often decided artificially and the improper choice may induce errors in quantitative intensity and phase retrieval. In order to obtain accurate in-focus information, quantitative intensity and phase imaging with the numerical focusing transport of intensity equation method combined with cellular duty ratio criterion and numerical wavefront propagation is introduced in this paper. Both numerical simulations and experimental measurements are provided proving this designed method can increase both retrieved in-focus intensity and phase accuracy and reduce dependence of focal plane determination in transport of intensity equation measurements. It is believed that the proposed method can be potentially applied in various fields as in-focus compensation for quantitative phase imaging and automatic focal plane determination, etc.

Much research effort has been spent in the 21st century on superresolution imaging techniques, methods which can beat the diffraction limit. Subwavelength composite structures called ``metamaterials" had initially shown great promise in superresolution imaging applications in the early 2000s, owing to their potential for nearly arbitrary capabilities in controlling light. However, for optical frequencies they are often plagued by absorption and scattering losses which can decay or destroy their interesting properties. Similar issues limit the application of other superresolution devices operating as effective media, or metal films that can transfer waves with large momentum by supporting surface plasmon polaritons. In this dissertation, new methods of mitigating the loss of object information in lossy and noisy optical imaging systems are presented. The result is an improvement in the upper bound on lateral spatial resolution. A concentration is placed on metamaterial and plasmonic imaging systems, and the same methods are subsequently adapted to more conventional far-field imaging systems. First, through numerical simulation it is shown that a lossy metamaterial lens has degraded imaging performance which can be partially compensated by deconvolution post-processing of the resultant image. This post-processing procedure is then shown to emulate a physical process called plasmon injection, which has been previously implemented to effectively remove the losses in a plasmonic metamaterial. Next, a more realistic scenario is considered; a thin film of silver acting as a near-field plasmonic ``superlens." In this case, methods are implemented to model incoherent light propagation so that the image can be reconstructed using only intensity data, removing the need for phase measurement. The same procedure from above is followed, and the resolution is enhanced. To push the resolution further, a spatial filtering method called active convolved illumination is developed to overcome the resolution limit set by the noise floor of the system. Finally, the spatial filtering methods are applied to more a more conventional far-field imaging system. Supported by experiment, the lateral resolution of a low numerical aperture imaging system is improved by blocking photons at the Fourier plane. For coherent light, a diffractive superlens is designed which uses the same principles from the above theory, except it encodes the high spatial frequency waves into propagating waves via a diffraction grating. The result is lateral resolution performance that surpasses similar previously published devices by 10 nm at a wavelength more than 80 nm longer.

Imaging systems using aspheric imaging lenses with complementary computation can deliver performance unobtainable in conventional imaging systems. These new imaging systems, termed Wavefront coded imaging systems, use specialized optics to capture a coded image of the scene. Decoding the intermediate image provides the "human-usable" image expected of an imaging system. Computation for the decoding step can be made completely transparent to the user with today's technology. Real-time Wavefront coded systems are feasible and cost-effective. This "computational imaging" technology can be adapted to solve a wide range of imaging problems. Solutions include the ability to provide focus-free imaging, to increase the field of view, to increase the depth of read, to correct for aberrations (even in single lens systems), and to account for assembly and temperature induced misalignment. Wavefront coded imaging has been demonstrated across a wide range of applications, including microscopy, miniature cameras, machine vision systems, infrared imaging systems and telescopes.

Optical microscopes are typically designed as versatile tools that are capable of imaging at various modalities with different resolutions in respective visual fields. A large extend of versatility causes increased complexity, size, and the cost. Imaging of tissue sections is a routine procedure in clinics and research laboratories. Tissue staining consumables, required time for staining along with the cost of the imaging systems are the limiting factors for widespread use. A multimode imaging system with enhanced resolution performance and automation is generally superfluous for the coarse resolution imaging of tissue sections. A dedicated system for label free tissue section imaging is presented. Label free imaging with high contrast is provided in form of quantitative phase. Earlier demonstrations of quantitative phase imaging of tissue sections utilized modified microscopes with different modalities for correlated imaging and motorized stages for field enlargement. Here, the system follows an off-axis digital holographic imaging configuration for the acquisition of quantitative phase at single shot. The system compromises from the resolution and magnification of a traditional microscope for the size of visual field, ease of use, and relative cost due to the exclusion of sample stages. A software level stitching further enlarges the effective field of view. Cite this article as: Toy MF. Quantitative Phase Macroscopic System for Label-Free Imaging of Tissue Sections. Electrica, 2021; 21(1): 10-19.

We present single-shot quantitative phase imaging with polarization differential interference contrast (PDIC) for a differential interference contrast microscope which records the unfiltered Stokes vector of the differential interference pattern with a polarization camera. PDIC enables high spatial resolution phase imaging in real-time, applicable to either absorptive or transparent samples and integrates simply with epi-fluorescence imaging. An algorithm with total variation regularization is further introduced to solve the quantitative phase map from the partial derivative along one single axis, improving the accuracy and the image quality from the Fourier transform solution. After quantifying the accuracy of PDIC phase imaging with numerical simulations and phantom measurements, we demonstrate the biomedical applications by imaging the quantitative phase of both stained and unstained histological tissue sections and visualizing the fission yeast Schizosaccharomyces pombe's cytokinesis.

The principle of equivalence between the imaging properties of scanning and conventional imaging systems has been proven in the past by assuming scalar sources of radiation and isotropic media. We prove here its general validity for optical systems with vector, electromagnetic sources of radiation and any optically nonactive media.

3D ultrasound imaging is becoming increasingly prevalent in the medical field. Compared to conventional 2D imaging systems, 3D imaging can provide a detailed view of tissue structures that makes diagnosis easier for the physicians. In addition, 2D image slices can be formed at various orientations to the transducer, making the examination less dependent on the skill of the sonographer. However, various challenges exist in developing a 3D imaging system, such as integration of a large number of elements, as well as post-processing of datasets received from a large number of channels. 2D transducer arrays are typically integrated with custom ICs in the probe handle to perform some intermediate beamforming and to reduce the number of cable connections to the imaging system. Capacitive micromachined ultrasonic transducers (CMUTs) have emerged as an alternative to piezoelectric transducers. Being a MEMS device, they greatly benefit from flexibility and ease of fabrication, and can be seamlessly integrated with electronics. Previous work demonstrates 3D stacking of CMUTs and dummy ICs with an intermediate interposer layer. However, that represents more of a mechanical demonstration of 3D integration. In this paper, we present a fully functional 3D ultrasound imaging system comprising a 32×32 2D CMUT array, 3D-stacked with front-end ICs using flip-chip bonding technology. The imaging system is capable of capturing real-time volumetric ultrasound data, and displaying 2D and 3D ultrasound images.

Conventional multichannel imaging systems comprise of many optical channels having similar imaging properties, namely field-of-view (FOV) and angular resolution/magnification. We demonstrated that the different optical channels can be designed such that each optical channel captures a different FOV and angular resolution compared to its neighboring channels. We designed and experimentally demonstrated a three-channel multiresolution imaging system where the first optical channel has the narrowest FOV (7°) and highest angular resolution (0.0096°) and the third optical channel has the widest FOV (80°) and lowest angular resolution (0.078°)1. The second optical channel has intermediate properties. The performance of the demonstrated three-channel imaging system however was affected by chromatic aberrations as it was designed for a single wavelength of 587.6 nm. The first optical channel was largely influenced by longitudinal chromatic aberration while the third channel by lateral chromatic aberration. Therefore, we have replaced the aspherical refractive lenses by hybrid lenses, which contain diffractive structures on top of their refractive surfaces, in the three-channel multiresolution imaging system to reduce its chromatic aberrations. The performance of the three channels with hybrid lenses is compared with those of the corresponding channels without hybrid lenses. The longitudinal color aberration of the first optical channel has been reduced from 1.7 mm to 0.2 mm; whereas the lateral color aberration of the third optical channel has been reduced from 250 μm to 14 μm. In conclusion, the hybrid lenses have reduced the chromatic aberrations of the three channels and extended the operating spectral range of the imaging system in the visible wavelength range.

We have designed and fabricated four LDA optical setups consisting of aberration compensated four different compact two hololens imaging systems. We have experimentally investigated and realized a hololens recording geometry which is interferogram of converging spherical wavefront with mutually coherent planar wavefront. Proposed real time monitoring and actual fringe field analysis techniques allow complete characterizations of fringes formed at measurement volume and permit to evaluate beam quality, alignment and fringe uniformity with greater precision. After experimentally analyzing the fringes formed at measurement volume by all four imaging systems, it is found that fringes obtained using compact two hololens imaging systems get improved both qualitatively and quantitatively compared to that obtained using conventional imaging system. Results indicate qualitative improvement of non-uniformity in fringe thickness and micro intensity variations perpendicular to the fringes, and quantitative improvement of 39.25% in overall average normalized standard deviations of fringe width formed by compact two hololens imaging systems compare to that of conventional imaging system.

Noninterferometric phase determination

Mammographic screening has played a pivotal role in reducing mortality rates associated with breast cancer. Despite an overall decline of breast cancer death rates of 43% from 1989 through 2020, breast cancer remains a predominant form of invasive cancer among women. Conventional X-ray imaging used in mammography can yield limited contrast when it comes to substances with similar X-ray attenuation, such as that between benign and cancerous tissue. Mesh-based X-ray phase imaging allows for higher contrast and resolution in similar-density materials. This thesis aims to explore characteristics of the mesh used in this kind of system.To investigate the significance of mesh orthogonality in mesh-based phase imaging, simulations were conducted. Three types of meshes were compared: an orthogonal mesh and oblique meshes with deviations from the horizontal or vertical axes. The use of mesh shifting demonstrated a significant enhancement in phase retrieval within the imaging systems that employed non-orthogonal meshes when compared to the trials conducted without mesh shifting. Next, two meshes of different compositions, stainless steel and nickel copper alloy meshes, were used in a biological phase imaging set-up. Theoretically, a higher-atomic-number mesh would produce a stronger absorption gradient, resulting in better phase images. However, noticeable, periodic artifacts were observed within the alloy mesh phase images that appeared rotated to the same degree as the mesh. Further investigation into other processing techniques might be advantageous to reduce artifacts.

Sparse synthetic transmit aperture (STA) imaging systems are a good alternative to the conventional phased array systems. Unfortunately, the sparse STA imaging systems suffer from some limitations, which can be overcome with a proper design. In order to do so, a simulated annealing algorithm, combined with an effective approach can used for optimization of a sparse STA ultrasound imaging system. In this paper, three two-stage algorithms for optimization of both the positions of the transmit sub-apertures and the weights of the receive elements are considered and studied. The first stage of the optimization employs a simulated annealing algorithm that optimizes the locations of the transmit sub-aperture centers for a set of weighting functions. Three optimization criteria used at this stage of optimization are studied and compared. The first two criteria are conventional. The third criterion, proposed in this paper, combines the first two criteria. At the second stage of optimization, an appropriate weighting function for the receive elements is selected. The sparse STA system under study employs a 64-element array, where all elements are used in receive and six sub-apertures are used in transmit. Compared to a conventional phased array imaging system, this system acquires images of better quality 21 times faster than an equivalent phased array system.