Modeling quantitative phase image formation under tilted illuminations

We present an optical metrology instrument for measuring both transmitted and reflected wavefront error (TWE and RWE) of coated or uncoated optics over a diameter of 5 inches. Depending on the coating transmittance and reflectance, the measurements have to be done at different wavelengths. Interferometer is a standard instrument to measure the TWE and RWE of uncoated optics. But in the case of coated optics (bandpass filters for example) measurement of TWE is not possible because the optics may not transmit the interferometer laser light. The chosen solution is based on a quadriwave lateral shearing interferometer (QWLSI) wavefront sensor. QWLSI is an achromatic technique, meaning that it measures OPD at any wavelength without any need for recalibration at specific wavelengths. Consequently, various sources at different wavelengths can be used with the same instrument and metrology bench. In addition, QWLSI measures the derivative of phase contrary to interferometer that measures phase. Therefore, QWLSI has by design a better WFE dynamic range for TWE and RWE measurement. Moreover accuracy (below 15nm RMS) and repeatability (below 2nm RMS) is perfectly adapted to optical metrology measurement. The optical solution is a standard double pass configuration composed of a collimator and a beam expander to adapt the size of the beam to the wavefront sensor aperture. We use LED sources to avoid any noise due to interferences within the optics, which occur with coherent light. We can use different wavelength between 400nm and 1100nm. We can optimize the longitudinal chromatic aberration by moving a lens from the beam expander. We characterized the bench according to the ISO 5725 standard for different wavelengths. Its precision was tested with different samples (filters and mirror). The precision on TWE was found to be below 2nm RMS.

Systematic error analysis and correction in quadriwave lateral shearing interferometer

The traditional multiposition method of the M2 factor measurement system is a good method, but it is relatively time consuming, so it cannot meet the requirements of the transient test of a Gaussian beam. To solve this problem, a quadriwave lateral shearing interferometer and a wavefront construction method based on a difference Zernike polynomial are analyzed. This interferometer uses a special grating to select four replicas of the wavefront, and the interferogram generated by four replicas includes difference wavefront information. Then the difference Zernike polynomial method is used to analyze quadriwave lateral shearing interferograms. The characteristic parameters are obtained after finding the optimal terms of the Zernike polynomials. As a result, the errors of F-number, beam radius, and radius of curvature are 3.7%, 3.8%, and 0.6%, respectively, which verifies this method to calculate parameters of Gaussian beam. In addition, we also find that the shear amount has influence on the reconstructed wavefront. It is showed that as the amount of shear increases from 20 pixels to 90 pixels, the peak-to-valley (P-V) values and RMS values both gradually decrease with a nonlinear relationship, which could be used to decrease the error of wavefront reconstruction further.

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.

Quantitative phase microscopy (QPM) is a label-free imaging technique often employed for long-term, high-contrast imaging of live bio-samples. Yet, QPM is not specific to a certain subcellular organelle. As a remedy, fluorescence microscopy can visualize specific subcellular organelles once being labeled with fluorescent markers. In this paper, a high-resolution phase/fluorescence dual-modality microscopic imaging method based on structured light illumination is proposed. In a dual-modality microscopic system, periodic stripes are generated by a digital micromirror array (DMD), and are used as the common illumination for both modalities. For QPM imaging, the holograms of the sample under structured light illuminations from different directions and phase shifts are recorded, from which a quantitative phase image with resolution enhancement can be reconstructed via a synthetic aperture procedure. Furthermore, a numerical approach is proposed to compensate for the environmental disturbances that often challenge aperture synthesis of phase imaging. This method determines each time the phase distortions caused by environmental disturbances through using the spectrum of the 0th order of the structured light illumination, and the phase distortions are removed from the phase distributions of the waves along the 0th and the ±1st diffraction orders. Resolution enhancement of QPM imaging is realized by synthesizing the spectra of all the waves along different diffraction orders of the structured light illuminations of different orientations. With phase images, three-dimensional shapes, inner structures, or refractive index distributions of transparent and translucent samples can be obtained. For fluorescence imaging, intensity images (morie patterns) of the sample under different structured light illuminations are recorded. The spectra along different diffraction orders are separated by using a phase shifting reconstruction algorithm, and are shifted to their original positions, forming a synthesized spectrum that is much broader than the spectra of raw intensity images (NA-limited spectra). An inverse Fourier transform on the synthesized spectrum yields a super-resolution fluorescence image of the sample. With the reconstructed fluorescence images, specific subcellular organelles labeled with fluorescent markers can be visualized. The combination of quantitative phase microscopy and fluorescence microscopy can obtain multidimensional information about the sample. In this dual-mode imaging system, the spatial resolution of quantitative phase imaging and fluorescence imaging are 840 nm and 440 nm, respectively. The proposed dual-mode microscopy imaging technique has been demonstrated for imaging fluorescent beads, fly wings, spring/rice leaves, mouse tail transection, and fluorescence-stained SiHa cells. We envisage that this method can be further applied to many fields, such as biomedicine, industry, and chemistry.

Optical diffraction tomography (ODT) is a promising label-free imaging method capable of quantitatively measuring the three-dimensional (3D) refractive index distribution of transparent samples. In recent years, partially coherent ODT (PC-ODT) has attracted increasing attention due to its system simplicity and absence of laser speckle noise. Quantitative phase imaging (QPI) technologies represented by Fourier ptychographic microscopy (FPM), differential phase contrast (DPC) imaging and intensity diffraction tomography (IDT) need to collect several or hundreds of intensity images, which usually introduce motion artifacts when shooting fast-moving targets, leading to a decrease in image quality. Hence, a quantitative real-time phase microscopy (qRPM) for extended depth of field (DOF) imaging based on 3D single-shot differential phase contrast (ssDPC) imaging method is proposed in this research study. qRPM incorporates a microlens array (MLA) to simultaneously collect spatial information and angular information. In subsequent optical information processing, a deconvolution method is used to obtain intensity stacks under different illumination angles in a raw light field image. Importing the obtained intensity stack into the 3D DPC imaging model is able to finally obtain the 3D refractive index distribution. The captured four-dimensional light field information enables the reconstruction of 3D information in a single snapshot and extending the DOF of qRPM. The imaging capability of the proposed qRPM system is experimental verified on different samples, achieve single-exposure 3D label-free imaging with an extended DOF for 160 µm which is nearly 30 times higher than the traditional microscope system.

A compact quadriwave lateral shearing interferometer (QWLSI) with strong adaptability and high precision is proposed based on a novel randomly encoded hybrid grating (REHG). By performing the inverse Fourier transform of the desired ±1 Fraunhofer diffraction orders, the amplitude and phase distributions of the ideally calculated quadriwave grating can be obtained. Then a phase chessboard is introduced to generate the same phase distribution, while the amplitude distribution can be achieved using the randomly encoding method by quantizing the radiant flux on the ideal quadriwave grating. As the Faunhofer diffraction of the REHG only contains the ±1 orders, no order selection mask is ever needed for the REHG-LSI. The simulations and the experiments show that the REHG-LSI exhibits strong adaptability, nice repeatability, and high precision.

Quantitative optical phase information provides an alternative method to observe biomedical properties, where conventional phase imaging fails. Phase retrieval typically requires multiple intensity measurements and iterative computations to ensure uniqueness and robustness against detection noise. To increase the measurement speed, we propose a single-shot quantitative phase imaging method with metasurface optics that can be conveniently integrated into conventional imaging systems with minimal modification. The improvement of the measurement speed is simultaneously made possible by combining deep learning with the transport-of-intensity equation. As a proof-of-concept, we demonstrate phase retrieval on both calibrated phase objects and biological specimens by using an imaging system integrated with our metasurface. When combined with the matched neural network, the system yields result with errors as low as 5% and increased space-bandwidth-product. A multitude of commercial applications can benefit from the compactness and rapid implementation of our proposed method.

Quantitative phase measurements at multiple wavelengths has created an opportunity for exploring new avenues in phase microscopy such as enhancing imaging-depth (1), measuring hemoglobin concentrations in erythrocytes (2), and more recently in tomographic mapping of the refractive index of live cells (3). To this end, quantitative phase imaging has been demonstrated both at few selected spectral points as well as with high spectral resolution (4,5). However, most of these developed techniques compromise imaging speed, field of view, or the spectral resolution to perform interferometric measurements at multiple colors. In the specific application of quantitative phase in studying blood diseases and red blood cells, current techniques lack the required sensitivity to quantify biological properties of interest at individual cell level. Recently, we have set out to develop a stable quantitative interferometric microscope allowing for measurements of such properties for red cells without compromising field of view or speed of the measurements. The feasibility of the approach will be initially demonstrated in measuring dispersion curves of known solutions, followed by measuring biological properties of red cells in sickle cell anemia.

With applications ranging from metabolomics to histopathology, quantitative phase microscopy (QPM) is a powerful label-free imaging modality. However, the speed of current QPM systems is limited by electronic hardware. To improve throughput further, here we propose differentiable optical-electronic quantitative phase microscopy (∂μ) that acquires images in a compressed form such that more information can be transferred beyond the electronic hardware bottleneck. The proposed microscopy uses optical feature extractors as image compressors. The resultant intensity distribution is then decompressed into QPM images by a reconstruction network. By optimizing optical-electronic parameters in an end-to-end manner, our method can improve the QPI throughput from Hours to Seconds (more than an order of magnitude).

Digital holographic microscopy (DHM) is an important technique that may be used for quantitative phase imaging of unstained biological cell samples. Since the DHM technology is not commonly used in clinics or bioscience research labs, at present there is no well-accepted focusing criterion for unstained samples that users can follow while recording image plane digital holograms of cells. The usual sharpness metrics that are useful for auto-focusing of stained cells do not work well for unstained cells as there is no amplitude contrast. In this work, we report a practical method for estimating the best focus plane for unstained cells in the digital hologram domain. The method is based on an interesting observation that for the best focus plane the fringe pattern associated with individual unstained cells predominantly shows phase modulation effect in the form of bending of fringes and minimal amplitude modulation. This criterion when applied to unstained red blood cells shows that the central dip in the doughnut-like phase profile of cells is maximal in this plane. The proposed methodology is helpful for standardizing the usage of DHM technology across different users and application developmentefforts. LAY DESCRIPTION: Digital holographic microscopy (DHM) is slowly but steadily becoming an important microscopy modality and gaining acceptability for basic bio-science research as well as clinical usage. One of the important features of DHM is that it allows users to perform quantitative imaging of unstained transparent cells. Instead of using dyes or fluorescent labelling, DHM systems use quantitative phase as a contrast mechanism which depends on the natural refractive index variation within the cell samples. Since minimal wet lab processing is required in order to image cell samples with a DHM, cells can be imaged in their natural state. While DHM is gaining popularity among users, the imaging protocols across the labs or users need to be standardized in order to make sure that the same quantitative phase parameters are used for tasks such as quantitative phased based cellclassification. One of the important operational tasks for any microscopy work is to focus the sample under study. While focusing comes naturally to users of brightfield microscopes based on image contrast, the focusing is not straightforward when samples are unstained so that they do not offer any amplitude contrast. When performing quantitative phase imaging, defocus can actually change the phase profile of the cell due to near-zone (Fresnel) diffraction effects. So unless a standardized focusing methodology is used, it will be difficult for multiple DHM users (potentially at different sites) to agree on quantitative results out of their phase images. DHM literature has prior works which perform numerical focusing of recovered complex wave-field in the hologram plane to find the best focus plane. However such methods are not user friendly and do not allow user the same focusing experience as in a brightfield microscope. The numerical focusing is therefore a reasonably good method for an optics researcher but not necessarily so for a microscopy technician looking at cell samples with a DHM system in a clinicalsetting. The present work provides a simple focusing criterion for unstained samples that works directly in the hologram domain. The technique is based on an interesting observation that the when an unstained cell sample is in the best-focus plane, its corresponding hologram (or fringe pattern) predominantly shows phase modulation manifested by bending of fringes at the location of the cell. This criterion can be converted into a simple numerical method as we have used to find the best-focus plane using a stack of through focus holograms. We believe that the technique can be used manually by visually observing the holograms or can be converted to an auto-focus algorithm for a motorized DHMsystem.

We have developed a modality for quantitative phase imaging within spectral domain optical coherence tomography based on using an off-axis reference beam. By tilting the propagation of the reference beam relative to that of the sample beam, a spatially varying fringe is generated. Upon detection of this fringe using a parallel spectral domain scheme, the fringe can be used to separate the interference component of the signal and obtain the complex sample field. In addition to providing quantitative phase measurements within a depth resolved measurement, this approach also allows elimination of the complex conjugate artifact, a known limitation of spectral interferometry. The principle of the approach is described here along with demonstration of its capabilities using technical samples.

We propose a lensfree on-chip microscopy approach for wide-field quantitative phase imaging (QPI) based on wavelength scanning. Unlike previous methods, we found that a relatively large-range wavelength diversity not only provides information to overcome spatial aliasing of the image sensor but also creates sufficient diffraction variations that can be used to achieve motion-free, pixel-super-resolved phase recovery. Based on an iterative phase retrieval and pixel-super-resolution technique, the proposed wavelength-scanning approach uses only eight undersampled holograms to achieve a half-pitch lateral resolution of 691 nm across a large field-of-view of 29.85mm2, surpassing 2.41 times the theoretical Nyquist-Shannon sampling resolution limit imposed by the pixel size of the sensor (1.67 µm). We confirmed the effectiveness of this technique in QPI and resolution enhancement by measuring the benchmark quantitative phase microscopy target. We also showed that this method can track HeLa cell growth within an incubator, revealing cellular morphologies and subcellular dynamics of a large cell population over an extended period of time.

Transmission optical coherence tomography (tOCT) is gaining interest for imaging biological samples; however, so far, quantitative phase information has not been directly obtained. This study introduces SOPHAST-OCT, an approach for integrated 2D phase recovery in Off-Axis Full-Field Swept-Source tOCT. It uses phase-shifting to leverage OCT’s inherent phase changes between recorded interferograms. SOPHAST-OCT demonstrates promising phase agreement with the simulated ground truth phase. This approach represents a first step toward quantitative imaging in tOCT. Full Text: PDF References Y. Park, C. Depeursinge, G. Popescu, "Quantitative phase imaging in biomedicine", Nature Photon 12, 578 (2018). CrossRef K. Alm, H. Cirenajwis, L. Gisselsson, A.G. Wingren, B. Janicke, A. Mölder, et al., in Holography, Research and Technologies (IntechOpen, 2011). CrossRef W. Drexler, J. Fujimoto, editors , Optical Coherence Tomography: Technology and Applications, 2nd ed. (Springer Cham, 2015). CrossRef W. Krauze, P. Ossowski, M. Nowakowski, M. Szkulmowski, M. Kujawińska, "Enhanced QPI functionality by combining OCT and ODT methods", Quantitative Phase Imaging VII, 11653, 19 (2021). CrossRef B.C. Wilson, S.L. Jacques, "Optical reflectance and transmittance of tissues: principles and applications", IEEE J. Quantum Electron. 26, 2186 (1990). CrossRef J. van der Horst, A.K. Trull, J. Kalkman, "Deep-tissue label-free quantitative optical tomography", Optica 7, 1682 (2020). CrossRef P. Hosseini, Y. Sung, Y. Choi, N. Lue, Z. Yaqoob, P. So, "Scanning color optical tomography (SCOT)", Opt. Expr. 23, 19752 (2015). CrossRef P. de Groot, "Phase Shifting Interferometry" in Optical Measurement of Surface Topography, edited by R. Leach (Springer, Berlin, Heidelberg, 2011). CrossRef M. Wojtkowski, A. Kowalczyk, R. Leitgeb, A.F. Fercher, "Full range complex spectral optical coherence tomography technique in eye imaging", Opt. Lett. 27, 1415 (2002). CrossRef Q. Zhang, S. Zhong, J. Lin, W. Chen, M. Luo, J. Zhong, Y. Yu, Z. Peng, S. Cheng, "Full-range Fourier-domain optical coherence tomography based on Mach–Zehnder interferometer", Opt. Lasers Engin. 124, 105794 (2020). CrossRef R. Juarez-Salazar, C. Robledo-Sanchez, F. Guerrero-Sanchez, A. Rangel-Huerta, "Generalized phase-shifting algorithm for inhomogeneous phase shift and spatio-temporal fringe visibility variation", Opt. Express 22, 4738 (2014). CrossRef R. Juarez-Salazar, C. Robledo-Sánchez, C. Meneses-Fabian, F. Guerrero-Sánchez, L. M. Arévalo Aguilar, "Generalized phase-shifting interferometry by parameter estimation with the least squares method", Opt. Lasers Engin. 51, 626 (2013). CrossRef M.A. Herráez, D.R. Burton, M.J. Lalor, M.A. Gdeisat, "Fast two-dimensional phase-unwrapping algorithm based on sorting by reliability following a noncontinuous path", Appl. Opt. 41, 7437 (2002). CrossRef Z. Wang, A.C. Bovik, "A universal image quality index", IEEE Signal Proc. Lett. 9, 81 (2002). CrossRef

Differential phase contrast (DPC) is a non-interferometric quantitative phase imaging method achieved by using an asymmetric imaging procedure. We report a pupil modulation differential phase contrast (PMDPC) imaging method by filtering a sample's Fourier domain with half-circle pupils. A phase gradient image is captured with each half-circle pupil, and a quantitative high resolution phase image is obtained after a deconvolution process with a minimum of two phase gradient images. Here, we introduce PMDPC quantitative phase image reconstruction algorithm and realize it experimentally in a 4f system with an SLM placed at the pupil plane. In our current experimental setup with the numerical aperture of 0.36, we obtain a quantitative phase image with a resolution of 1.73μm after computationally removing system aberrations and refocusing. We also extend the depth of field digitally by 20 times to ±50μm with a resolution of 1.76μm.