Phase Imaging Technique Research Articles

Investigating the Joint Amplitude and Phase Imaging of Stained Samples in Automatic Diagnosis.

The diagnosis of many diseases relies, at least on first intention, on an analysis of blood smears acquired with a microscope. However, image quality is often insufficient for the automation of such processing. A promising improvement concerns the acquisition of enriched information on samples. In particular, Quantitative Phase Imaging (QPI) techniques, which allow the digitization of the phase in complement to the intensity, are attracting growing interest. Such imaging allows the exploration of transparent objects not visible in the intensity image using the phase image only. Another direction proposes using stained images to reveal some characteristics of the cells in the intensity image; in this case, the phase information is not exploited. In this paper, we question the interest of using the bi-modal information brought by intensity and phase in a QPI acquisition when the samples are stained. We consider the problem of detecting parasitized red blood cells for diagnosing malaria from stained blood smears using a Deep Neural Network (DNN). Fourier Ptychographic Microscopy (FPM) is used as the computational microscopy framework to produce QPI images. We show that the bi-modal information enhances the detection performance by 4% compared to the intensity image only when the convolution in the DNN is implemented through a complex-based formalism. This proves that the DNN can benefit from the bi-modal enhanced information. We conjecture that these results should extend to other applications processed through QPI acquisition.

Single-shot blind deconvolution in coherent diffraction imaging with coded aperture

We present a method for single-shot blind deconvolution in coherent diffraction imaging. Coherent diffraction imaging is a technique for non-interferometric quantitative phase imaging without reference light. In our method based on coherent diffraction imaging, a complex amplitude object is illuminated with coherent light, and light from the object is captured through unknown aberrating media and a coded aperture located on the pupil plane to reduce estimated variables on the aberrated pupil function. Both the amplitude and the phase of the object are recovered from the single captured intensity image by a phase retrieval algorithm in which the coded aperture is utilized as a support to estimate the sparse aberrated pupil function. We numerically and experimentally demonstrate the proposed method with complex amplitude objects under severe aberrating conditions. In the experiment, we quantitatively evaluate its performance with ptychography, which is a method for multi-shot coherent diffraction imaging. Our method enables quantitative phase imaging through turbulence by using simple and reference-free optical hardware without any invasive process.

Accurate dynamic quantitative phase imaging using multi-wavelength multiplexing

We present a novel, accurate, full-filed, dynamic quantitative phase imaging (QPI) technique by using multi-wavelength multiplexing and multi-plane iterative phase retrieval algorithm. In our method, a liquid crystal spatial light modulator is employed to flexibly generate multiple defocus intensity images at once, using its adjustable phase modulation characteristics of different wavelengths. Then these images contained at different wavelengths are captured by two color cameras with single exposure. To achieve accurate QPI, a multi-plane iterative phase reconstruction algorithm is also proposed based on transport of intensity equation (TIE). Finally, with these multiple defocus images, an accurate dynamic phase result can be provided by our approach. In addition, the errors caused by color coupling of color camera and chromatic aberration of the optical system are both analyzed and effectively compensated. Experiments conducted on the phase plate, living human colorectal cancer cells and human red blood cells well demonstrate the accuracy, dynamic measurement ability and flexibility of our method.

Video-rate quantitative phase imaging with dynamic acousto-optic defocusing

Various quantitative phase imaging techniques exist capable of characterizing transparent and low-contrast samples without the addition of dyes or fluorescent probes. Among them, the transport of intensity equation (TIE) allows phase retrieval by capturing information from different focal planes without complex inteferometric setups. However, current implementations can be limited in speed or accuracy by the lack of optical systems suitable for fast, reliable, and customizable focal plane selection. Here, we report how combining acousto-optics with pulsed illumination enables accurate and on-demand electronic defocus control suitable for TIE phase imaging at speeds only limited by the camera frame rate. The system exhibits diffraction-limited spatial resolution and high reconstruction fidelity, undistinguishable from traditional mechanical defocusing. We demonstrate its feasibility by measuring different dynamic events at rates as high as 100 phase maps per second. The tunability and ease of implementation of our system can pave the way to democratizing quantitative phase imaging in histopathology, fluid dynamics, and other fields involving thin transparent samples.

PtyLab.m/py/jl: a cross-platform, open-source inverse modeling toolbox for conventional and Fourier ptychography.

Conventional (CP) and Fourier (FP) ptychography have emerged as versatile quantitative phase imaging techniques. While the main application cases for each technique are different, namely lens-less short wavelength imaging for CP and lens-based visible light imaging for FP, both methods share a common algorithmic ground. CP and FP have in part independently evolved to include experimentally robust forward models and inversion techniques. This separation has resulted in a plethora of algorithmic extensions, some of which have not crossed the boundary from one modality to the other. Here, we present an open source, cross-platform software, called PtyLab, enabling both CP and FP data analysis in a unified framework. With this framework, we aim to facilitate and accelerate cross-pollination between the two techniques. Moreover, the availability in Matlab, Python, and Julia will set a low barrier to enter each field.

Quantitative phase imaging comparison of digital holographic microscopy and transport of intensity equation phase through simultaneous measurements of live cells

Quantitative phase imaging (QPI) of live cells allows label-free assessment of cell morphology and quantitative estimation of cell thickness, and therefore volume. Of the techniques available for QPI, digital holographic microscopy (DHM) is an accurate and reliable high-resolution phase imaging technique, but assembly and operation of a DHM capable setup can require significant optical engineering experience and specialized equipment that may not be practical for many labs. Transport of intensity equation (TIE) imaging is a QPI method that is compatible with conventional bright field microscopy, has a lower implementation cost without the need for specialized optics or alignment, and has significantly reduced spatial and temporal coherence requirements when compared to DHM. However, TIE is susceptible to different forms of noise than DHM and its accuracy in live cell imaging has not been well studied. Here, we present a microscope capable of performing simultaneous DHM and TIE phase reconstructions to compare both methods and validate TIE as an accurate phase retrieval method both for a static sample array and for live cells.

Multimodal study of CHI3L1 inhibition and its effect on angiogenesis, migration, immune response and refractive index of cellular structures in glioblastoma

Glioblastoma is one of the most aggressive tumours with a poor response to treatment and a poor prognosis for patients. One of the proteins expressed in glioblastoma tissue is CHI3L1 (YKL-40), which is upregulated and known for its angiogenesis-supporting and pro-tumour immunomodulatory effects in a variety of cancers. In this paper we present the anti-angiogenic, anti-migratory and immunomodulatory effects of the compound G721–0282, an inhibitor of CHI3L1. The inhibitor-induced changes were investigated using conventional techniques as well as the novel label-free digital holographic tomography (DHT), a quantitative phase imaging technique that allows the reconstruction of the refractive index (RI), which is used as an image contrast for 3D visualisation of living cells. DHT allowed digital staining of individual cells and intercellular structures based only on their specific RI. Quantitative spatially resolved analysis of the RI data shows that the concentration of G721–0282 leads to significant changes in the density of cells and their intracellular structures (in particular the cytoplasm and nucleus), in the volume of lipid droplets and in protein concentrations. Studies in the U-87 MG glioblastoma cell line, THP-1 monocytes differentiated into macrophages, human microvascular endothelial cells (HMEC-1) and in the spheroid model of glioblastoma composed of U-87 MG, HMEC-1 and macrophages suggest that inhibition of CHI3L1 may have potential in the antitumour treatment of glioblastoma. In this paper, we also propose a spheroid model for in vitro studies that mimics this type of tumour.

Towards true volumetric refractive index investigation in tomographic phase microscopy at the cellular level

Tomographic phase microscopy (TPM) is one of the most widely used quantitative phase imaging techniques. It is a non-invasive, label-free technique that allows high-resolution imaging. It enables the morphology of a living cell to be captured without chemical treatment, by using the refractive index (RI) as a contrast parameter to image internal structures. In this paper, we demonstrate that due to the fact that biological cells are highly heterogeneous structures, it is crucial to use full volumetric data to calculate the average RI values of biological samples and their organelles. To prove our point, we present tomographic reconstructions of 3 cell types: neuroblastoma SH-SY5Y, adenocarcinoma A549 and leukemia HL-60, from which we calculate the average RI value from the 3D volumetric data and compare it to the average RI values calculated from each 2D section.

Fabrication of High‐Aspect Ratio Nanogratings for Phase‐Based X‐Ray Imaging

AbstractDiffractive optical elements such as periodic gratings are fundamental devices in X‐ray imaging – a technique that medical, material science, and security scans rely upon. Fabrication of such structures with high aspect ratios at the nanoscale creates opportunities to further advance such applications, especially in terms of relaxing X‐ray source coherence requirements. This is because typical grating‐based X‐ray phase imaging techniques (e.g., Talbot self‐imaging) require a coherence length of at least one grating period and ideally longer. In this paper, the fabrication challenges in achieving high‐aspect ratio nanogratings filled with gold are addressed by a combination of laser interference and nanoimprint lithography, physical vapor deposition, metal assisted chemical etching (MACE), and electroplating. This relatively simple and cost‐efficient approach is unlocked by an innovative post‐MACE drying step with hexamethyldisilazane, which effectively minimizes the stiction of the nanostructures. The theoretical limits of the approach are discussed and, experimentally, X‐ray nanogratings with aspect ratios >40 are demonstrated. Finally, their excellent diffractive abilities are shown when exposed to a hard (12.2 keV) monochromatic X‐ray beam at a synchrotron facility, and thus potential applicability in phase‐based X‐ray imaging.

The Near Infrared Imager and Slitless Spectrograph for the James Webb Space Telescope. IV. Aperture Masking Interferometry

The James Webb Space Telescope’s Near Infrared Imager and Slitless Spectrograph (JWST-NIRISS) flies a 7-hole non-redundant mask (NRM), the first such interferometer in space, operating at 3–5 μm wavelengths, and a bright limit of ≃4 mag in W2. We describe the NIRISS Aperture Masking Interferometry (AMI) mode to help potential observers understand its underlying principles, present some sample science cases, explain its operational observing strategies, indicate how AMI proposals can be developed with data simulations, and how AMI data can be analyzed. We also present key results from commissioning AMI. Since the allied Kernel Phase Imaging (KPI) technique benefits from AMI operational strategies, we also cover NIRISS KPI methods and analysis techniques, including a new user-friendly KPI pipeline. The NIRISS KPI bright limit is ≃8 W2 (4.6 μm) magnitudes. AMI NRM and KPI achieve an inner working angle of ∼70 mas, which is well inside the ∼400 mas NIRCam inner working angle for its circular occulter coronagraphs at comparable wavelengths.

White blood cell detection, classification and analysis using phase imaging with computational specificity (PICS)

Treatment of blood smears with Wright’s stain is one of the most helpful tools in detecting white blood cell abnormalities. However, to diagnose leukocyte disorders, a clinical pathologist must perform a tedious, manual process of locating and identifying individual cells. Furthermore, the staining procedure requires considerable preparation time and clinical infrastructure, which is incompatible with point-of-care diagnosis. Thus, rapid and automated evaluations of unlabeled blood smears are highly desirable. In this study, we used color spatial light interference microcopy (cSLIM), a highly sensitive quantitative phase imaging (QPI) technique, coupled with deep learning tools, to localize, classify and segment white blood cells (WBCs) in blood smears. The concept of combining QPI label-free data with AI for the purpose of extracting cellular specificity has recently been introduced in the context of fluorescence imaging as phase imaging with computational specificity (PICS). We employed AI models to first translate SLIM images into brightfield micrographs, then ran parallel tasks of locating and labelling cells using EfficientNet, which is an object detection model. Next, WBC binary masks were created using U-net, a convolutional neural network that performs precise segmentation. After training on digitally stained brightfield images of blood smears with WBCs, we achieved a mean average precision of 75% for localizing and classifying neutrophils, eosinophils, lymphocytes, and monocytes, and an average pixel-wise majority-voting F1 score of 80% for determining the cell class from semantic segmentation maps. Therefore, PICS renders and analyzes synthetically stained blood smears rapidly, at a reduced cost of sample preparation, providing quantitative clinical information.

Stain-free identification of cell nuclei using tomographic phase microscopy in flow cytometry

Quantitative phase imaging has gained popularity in bioimaging because it can avoid the need for cell staining, which, in some cases, is difficult or impossible. However, as a result, quantitative phase imaging does not provide the labelling of various specific intracellular structures. Here we show a novel computational segmentation method based on statistical inference that makes it possible for quantitative phase imaging techniques to identify the cell nucleus. We demonstrate the approach with refractive index tomograms of stain-free cells reconstructed using tomographic phase microscopy in the flow cytometry mode. In particular, by means of numerical simulations and two cancer cell lines, we demonstrate that the nucleus can be accurately distinguished within the stain-free tomograms. We show that our experimental results are consistent with confocal fluorescence microscopy data and microfluidic cyto-fluorimeter outputs. This is a remarkable step towards directly extracting specific three-dimensional intracellular structures from the phase contrast data in a typical flow cytometry configuration.

(Digital Presentation) Investigation of Microstructure Dependent Inhibitor Adsorption Mechanism on Carbon Steel By in Situ Atomic Force Microscopy

In the petroleum industry, organic surfactant type inhibitors have been widely applied in the oil and gas industry considering their high efficiency. Carbon steels with various carbide contents have also been used extensively as pipeline materials during the transport and storage of crude oil due to their excellent mechanical properties and low cost. The previous investigation of inhibition mechanism on carbon steel is limited by lack of surface characterization techniques at a molecular level. Atomic force microscopy (AFM) can not only provide localized visual observation, but can also achieve the detection of mechanical properties of an inhibitor film. In this study, in situ AFM contact mode friction imaging and in situ AFM tapping mode phase imaging techniques have been applied to investigate the influence of the ferritic-pearlitic microstructure of carbon steel on inhibitor adsorption mechanisms during CO2 corrosion under different inhibitor concentrations. At concentration above surface saturation, AFM contact mode friction images show a large friction contrast between inhibitor covered cementite structures and ferrite structures, while in the absence of inhibitor, this friction contrast almost disappears, indicating the inhibitor adsorption induced this difference. AFM tapping mode phase images indicate uniform adsorption of inhibitor on both cementite and ferrite structures.The differences observed in these AFM analyses indicate that the adhesion force of inhibitor molecules on cementite could be smaller than on ferrite, or the molecular orientations of inhibitor molecules adsorbed on cementite and ferrite structures could be different. The special adsorption behavior of inhibitor molecules on cementite means the carbide component of the microstructure could directly influence inhibitor adsorption and may decrease inhibitor efficiency. Simultaneous linear polarization resistance tests in electrochemical AFM (EC-AFM) cell indicates a maximum inhibition efficiency at concentration above saturation.Keywords : Contact mode AFM, friction contrast, tapping mode AFM, phase contrast, cementite, ferrite, organic inhibitor, adhesion force.Statement of importance:Studies related to inhibitor adsorption mechanisms on ferritc-pearlitic carbon steels can be connected with the practical issues that iron carbide can impair the corrosion inhibitor performance on carbon steels. This AFM work connects the corrosion inhibition effect with the carbon steel components at a molecular level for the first time. Physical explanations regarding the interactions between inhibitor molecules and carbon steel microstructures can also be provided with the assistance of molecular simulations.Figure caption: Contact and tapping mode AFM images of inhibitor film formed on UNS G1018 steel at 2 CMC (100ppm) tetradecyldimethylbenzylammonium inhibitor + 1 wt% NaCl. The contact mode friction image indicates a large friction contrast between inhibitor covered cementite and ferrite structure, while the tapping mode phase image indicates a negligible softness contrast between cementite and ferrite structure. Figure 1

Single-shot quantitative phase imaging with phase modulation of a liquid crystal spatial light modulator (LC-SLM) under white light illumination.

We propose a novel, to the best of our knowledge, single-shot quantitative phase imaging (QPI) technique with the phase modulation of a liquid crystal spatial light modulator (LC-SLM) under white light illumination. By studying the phase modulation characteristics of an LC-SLM under white light illumination, images captured at different wavelengths are equivalent to those captured at different defocus distances when loading a Fresnel lens pattern on the LC-SLM. Consequently, a color camera is able to simultaneously acquire multi-intensity images at different defocus distances. Finally, the phase is retrieved from a single-shot color image using the transport of intensity equation. To demonstrate the flexibility and accuracy of our method, a photoetched phase object and human red blood cells are quantitatively measured. An investigation of living yeast cells is conducted to verify the dynamic measurement capability. The proposed method provides a simple, efficient, and flexible means to accomplish real-time high-resolution quantitative phase imaging without sacrificing the field of view (FOV), which can be further integrated into a conventional microscope to achieve real-time microscopic QPI.

Simple phase unwrapping method with continuous convex minimization.

Phase unwrapping is a problem to reconstruct true phase values from modulo 2π phase values measured using various phase imaging techniques. This procedure is essentially formulated as a discrete optimization problem. However, most energy minimization methods using continuous optimization techniques have ignored the discrete nature and solved it as a continuous minimization problem directly, leading to losing exactness of the algorithms. We propose a new minimum norm method that can yield the optimal solution of the discrete problem by minimizing a continuous energy function. In contrast to the graph-cuts method, which is state of the art in this field, the proposed method requires much less memory space and a very simple implementation. Therefore, it can be simply extended to 3D or 4D phase unwrapping problems.

Evaluation of late arterial acquisition and image quality after gadoxetate disodium injection using the CDT-VIBE sequence

To explore the applicability of multi-arterial phase imaging technique in gadoxetate disodium-enhanced MRI. We studied 140 consecutive patients with suspected liver lesions who underwent gadoxetate disodium-enhanced MRI before surgery. All patients were randomized into three groups: group A (n = 50) was examined with VIBE-based single-artery phase imaging, group B (n = 44) with StarVIBE, and group C (n = 46) with CAIPIRINHA-Dixon-TWIST-VIBE (CDT-VIBE)-based multi-artery phase imaging. We evaluated the display rate of late arterial images and image quality in arterial phase images. We performed a study of 140 consecutive patients suspected with liver lesions who received gadoxetate disodium-enhanced MRI examination before surgery. All patients were randomly divided into three groups: group A (n = 50) was examined with single arterial phase imaging based on VIBE, group B (n = 44) was based on StarVIBE and group C (n = 46) was analyzed with multi-arterial phase imaging based on CAIPIRINHA-Dixon-TWIST-VIBE (CDT-VIBE). We evaluated the display rate of late arterial images and the image quality of dynamically enhanced images. Both radiologists had an almost perfect agreement (Kappa value > 0.8) in the assessment of late arterial and image quality. For late arterial acquisition, group C was superior to groups A and B (x2 = 18.940, P < 0.05); The image of phase 4 had the highest display rate in the late artery phase. For arterial phase image quality, there was no difference between groups A, B and C at five phases (H = 10.481, P = 0.106); and the best image quality score was lower in group C than in groups A and B (H = 8.573, P = 0.014).For the quality of the late arterial images, there was a statistical difference between the best images in groups A, B and C (H = 6.619, P = 0.037), and the images in group C were significantly better than those in group A (P.adj < 0.05). By applying multi-arterial phase acquisition based on CDT-VIBE, gadoxetate disodium-enhanced MRI scanning can obtain a better late arterial phase and provide high-quality images with fewer motion artifacts.

Natural-like Chalcones with Antitumor Activity on Human MG63 Osteosarcoma Cells.

Osteosarcoma (OS) is a malignant disease characterized by poor prognosis due to a high incidence of metastasis and chemoresistance. Recently, Licochalcone A (Lic-A) has been reported as a promising agent against OS. Starting from chalcones selected from a wide in-house library, a new series was designed and synthetized. The antitumor activity of the compounds was tested on the MG63 OS cell line through the innovative Quantitative Phase Imaging technique and MTT assay. To further investigate the biological profile of active derivatives, cell cycle progression and apoptosis induction were evaluated. An earlier and more consistent arrest in the G2-M phase with respect to Lic-A was observed. Moreover, apoptosis was assessed by Annexin V staining as well as by the detection of typical morphological features of apoptotic cells. Among the selected compounds, 1e, 1q, and 1r proved to be the most promising antitumor molecules. This study pointed out that an integrated methodological approach may constitute a valuable platform for the rapid screening of large series of compounds.

Holographic point source for digital lensless holographic microscopy.

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.

Model-based feedback control for on-demand droplet dispensing system with precise real-time phase imaging

Recent years have witnessed the advances of droplet-based microfluidics as a powerful tool for high-throughput assays in biology, chemistry, material and environmental sciences. Precise and effective droplet generation is critical for the practical applications of droplet-based microfluidics. Although many passive and active droplet generation methods have been proposed and demonstrated, there are still challenges towards the implementation of droplet-on-demand in terms of system complexity, precision and robustness. In this work, we propose and demonstrate a dual-pressure-pulse (DPP) driven droplet generation method incorporated with model-based feedback control to achieve on-demand droplet generation. Based on the study of droplet formation dynamics in a flow-focusing configuration using real-time quantitative phase imaging (QPI) technique, we validate the proposed dual-pressure-pulse (DPP) driven droplet generation concept, for which the physical model of droplet formation can be greatly simplified. With this approach, model-based feedback control can be implemented for DPP actuation to achieve on-demand droplet generation with relatively high system response and low steady-state error.

Scanning-free digital holography for decoupling the refractive index and thickness via a constrained equation of higher degree.

Digital holography is one of the most popular quantitative phase imaging techniques, but the refractive index and the thickness are always coupled in the phase. To solve the decoupling problem, multiple scanning methods such as tomography and total reflection are usually used, which is time-consuming. To increase the imaging speed and reduce the system cost, it is urgent to seek the decoupling method of scanning-free digital holography. In this paper, we find that the decoupling method of scanning-free digital holography can be transformed into a problem of solving constrained higher order equations. By introducing the Fresnel reflection formula, a six-degree equation about refractive index is constructed and the corresponding algorithm for solving the equation is given. By using the algorithm, the refractive index and thickness can be decoupled successfully. A series of results show that the proposed method is effective and has high anti-noise performance. This method provides a mathematical possibility for scanning-free digital holography to decouple the refractive index and complex pixel stepped thickness distributions. Therefore, it may provide a theoretical basis for the subsequent development of a real scanning-free digital holography system, which may have potential applications in the measurement of optical devices produced by the modern film deposition process and etching process.