Quantitative Phase Imaging Techniques Research Articles

Quantitative phase imaging techniques for measuring scattering properties of cells and tissues: a review-part I.

Quantitative phase imaging (QPI) techniques offer intrinsic information about the sample of interest in a label-free, noninvasive manner and have an enormous potential for wide biomedical applications with negligible perturbations to the natural state of the sample in vitro. We aim to present an in-depth review of the scattering formulation of light-matter interactions as applied to biological samples such as cells and tissues, discuss the relevant quantitative phase measurement techniques, and present a summary of various reported applications. We start with scattering theory and scattering properties of biological samples followed by an exploration of various microscopy configurations for 2D QPI for measurement of structure and dynamics. We reviewed 157 publications and presented a range of QPI techniques and discussed suitable applications for each. We also presented the theoretical frameworks for phase reconstruction associated with the discussed techniques and highlighted their domains of validity. We provide detailed theoretical as well as system-level information for a wide range of QPI techniques. Our study can serve as a guideline for new researchers looking for an exhaustive literature review of QPI methods and relevant applications.

Birefringence mapping of biological tissues based on polarization sensitive non-interferometric quantitative phase imaging technique

ObjectiveOral cancer is a leading cause of mortality globally, particularly affecting developing regions where oral hygiene is often overlooked. The optical properties of tissues are vital for diagnostics, with polarization imaging emerging as a label-free, contrast-enhancing technique widely employed in medical and scientific research over past few decades. Materials and methodsWe present a novel polarization sensitive quantitative phase imaging of biological tissues by incorporating the conventional polarization microscope and transport of intensity equation-based phase retrieval algorithm. This integration provides access to the birefringence mapping of biological tissues. The inherent optical anisotropy in biological tissues induces the polarization dependent refractive index variations which can provide the detailed insights into the birefringence characteristics of their extracellular constituents. Experimental investigations were conducted on both normal and cancerous oral tissue samples by recording a set of three polarization intensity images for each case with a step size of 2 μm. ResultsA noteworthy increment in birefringence quantification was observed in cancerous as compared to the normal tissues, attributed to the proliferation of abnormal cells during cancer progression.The mean birefringence values were calculated for both normal and cancerous tissues, revealing a significant increase in birefringence of cancerous tissues (2.1 ± 0.2) × 10−2 compared to normal tissues (0.8 ± 0.2) × 10−2. Data were collected from 8 patients in each group under identical experimental conditions. ConclusionThis polarization sensitive non-interferometric optical approach demonstrated effective discrimination between cancerous and normal tissues, with various parameters indicating elevated values in cancerous tissues.

EVATOM: an optical, label-free, machine learning assisted embryo health assessment tool

The combination of a good quality embryo and proper maternal health factors promise higher chances of a successful in vitro fertilization (IVF) procedure leading to clinical pregnancy and live birth. Of these two factors, selection of a good embryo is a controllable aspect. The current gold standard in clinical practice is visual assessment of an embryo based on its morphological appearance by trained embryologists. More recently, machine learning has been incorporated into embryo selection “packages”. Here, we report EVATOM: a machine-learning assisted embryo health assessment tool utilizing an optical quantitative phase imaging technique called artificial confocal microscopy (ACM). We present a label-free nucleus detection method with, to the best of our knowledge, novel quantitative embryo health biomarkers. Two viability assessment models are presented for grading embryos into two classes: healthy/intermediate (H/I) or sick (S) class. The models achieve a weighted F1 score of 1.0 and 0.99 respectively on the in-distribution test set of 72 fixed embryos and a weighted F1 score of 0.9 and 0.95 respectively on the out-of-distribution test dataset of 19 time-instances from 8 live embryos.

Recent Advances and Current Trends in Transmission Tomographic Diffraction Microscopy.

Optical microscopy techniques are among the most used methods in biomedical sample characterization. In their more advanced realization, optical microscopes demonstrate resolution down to the nanometric scale. These methods rely on the use of fluorescent sample labeling in order to break the diffraction limit. However, fluorescent molecules' phototoxicity or photobleaching is not always compatible with the investigated samples. To overcome this limitation, quantitative phase imaging techniques have been proposed. Among these, holographic imaging has demonstrated its ability to image living microscopic samples without staining. However, for a 3D assessment of samples, tomographic acquisitions are needed. Tomographic Diffraction Microscopy (TDM) combines holographic acquisitions with tomographic reconstructions. Relying on a 3D synthetic aperture process, TDM allows for 3D quantitative measurements of the complex refractive index of the investigated sample. Since its initial proposition by Emil Wolf in 1969, the concept of TDM has found a lot of applications and has become one of the hot topics in biomedical imaging. This review focuses on recent achievements in TDM development. Current trends and perspectives of the technique are also discussed.

Comparative analysis of digital holographic microscopy and digital lensless holographic microscopy for quantitative phase imaging

This study provides a detailed comparison of two widely used quantitative phase imaging (QPI) techniques: single-shot off-axis digital holographic microscopy (DHM) and digital lensless holographic microscopy (DLHM). The primary aim is to evaluate and contrast critical aspects of their imaging performance, including spatial phase sensitivity, phase measurement accuracy, and spatial lateral resolution. Employing typical configurations for both DHM and DLHM, the study utilizes a customized phase test target featuring linear phase changes introduced by a specially designed linear density attenuation filter. Ground truth data from an atomic force microscope is incorporated to validate the experimental findings. The comparative analysis reveals that DHM and DLHM exhibit nearly identical spatial phase sensitivity, with DHM demonstrating a minimal 3.2% measurement error compared to DLHM's 4% in height measurement accuracy. Notably, DHM achieves a finer spatial lateral resolution down to 3.1 µm, surpassing DLHM's 5.52 µm. While DHM outperforms DLHM in precision and resolution, the latter offers advantages in terms of portability and cost-effectiveness. These findings provide valuable insights for researchers and practitioners, aiding in the informed selection of QPI methods based on specific application requirements.

Common-path digital holographic microscopy based on a volume holographic grating for quantitative phase imaging.

Digital holographic microscopy (DHM) is a powerful quantitative phase imaging (QPI) technique that is capable of recording sample's phase information to enhance image contrast. In off-axis DHM, high-quality QPI images can be generated within a single recorded hologram, and the system stability can be enhanced by common-path configuration. Diffraction gratings are widely used components in common-path DHM systems; however, the presence of multiple diffraction beams leads to system power loss. Here, we propose and demonstrate implementation of a volume holographic grating (VHG) in common-path DHM, which provides single diffraction order. VHG in common-path DHM (i.e., VHG-DHM) helps in improving signal-to-noise ratio as compared to the conventional DHM. In addition, VHG, with inherently high angular selectivity, reduces image noise caused by stray light. With a simple fabrication process, it is convenient to utilize VHG to control the beam separation angle of DHM. Further, by using Bragg-matched wavelength degeneracy to avoid potential cell damaging effect in blue light, the VHG is designed for recording at a maximum sensitive wavelength of ∼488 nm, while our VHG-DHM is operated at the longer wavelength of red 632.8 nm for cell observation. Experimental results, measured by the VHG-DHM, show the measurement of target thickness ranging from 100 nm to 350 nm. In addition, stability of the system is quantitatively measured. High-contrast QPI images of human lung cancer cells are demonstrated.

Leveraging the depolarization of scattered light for holography with the Stokes correlation

A correlation holography technique for quantitative phase imaging from scattered depolarized light is proposed and experimentally demonstrated. To implement this technique, we exploit the depolarization of scattered light with the higher-order Stokes correlation. The higher-order Stokes correlation along with the two-step phase-shifting method is utilized to retrieve the complex Fourier coefficient, this subsequently supports the recovery of the phase in the correlation holography with the fourth-order correlation. A detailed theoretical framework is developed and is also supported by numerical simulation and experimental tests. The applicability of the proposed technique is experimentally demonstrated by quantitatively reconstructing different phase objects from the scattered depolarized light.

Single-image phase retrieval for off-the-shelf Zernike phase-contrast microscopes.

Quantitative phase imaging (QPI), such as digital holography, is considered a promising tool in the field of life science due to its noninvasive and quantitative visualization capabilities without the need for fluorescence labeling. However, the popularity of QPI systems is limited due to the cost and complexity of their hardware. In contrast, Zernike phase-contrast microscopy (ZPM) has been widely used in practical scenarios but has not been categorized as QPI, owing to halo and shade-off artifacts and the weak phase condition. Here, we present a single-image phase retrieval method for ZPM that addresses these issues without requiring hardware modifications. By employing a rigorous physical model of ZPM and a gradient descent algorithm for its inversion, we achieve single-shot QPI with an off-the-shelf ZPM system. Our approach is validated in simulations and experiments, demonstrating QPI of a polymer microbead and biological cells. The quantitative nature of our method for single-cell imaging is confirmed through comparisons with observations from an established QPI technique conducted through digital holography. This study paves the way for transforming non-QPI ZPM systems into QPI systems.

#264 : 3D Time-Lapse Refractive-Index Imaging of Intact Mouse Embryos for Non-Invasive Evaluation of the Embryonic Development

Background and Aims: Time-lapse technology is an emerging assisted reproductive technology (ART) that enables simultaneous culture and monitoring of embryos in vitro during their development. It provides extra information on key events during embryo development, which can assist embryologists in selecting embryos alongside traditional morphology evaluations. Various imaging technologies have been utilised to visualise and evaluate embryos such as brightfield microscopy, darkfield microscopy, and Hoffman modulation contrast microscopy. Although these imaging technologies allow for non-invasive imaging of human embryos in a clinical setting, 3D imaging of a whole embryo with subcellular resolution remains a challenge. Method: We demonstrate 3D time-lapse label-free imaging of live mouse embryos using the 3D refractive index (RI) imaging technique. The proposed method is a quantitative phase imaging technique that measures multiple-intensity images of transmitted light through the embryos via axial scanning to reconstruct the 3D RI map of the embryos. Results: Using the proposed method, live mouse embryos were monitored at different developmental stages, ranging from 2-cell embryos to expanded blastocysts. The measured 3D RI maps of the developing embryos reveal 3D landscape of subcellular features inside the embryos (Fig. 1). In the quantitative assessment of embryonic development, various morphokinematic parameters were extracted from the measured 3D RI maps of the embryos. Conclusion: Based on the results, we envision that our method has broad applications where non-invasive and quantitative assessment of embryonic development is required.

Development of Robust On-Demand Droplet Generation System Using 3-D Image Reconstruction as Feedback

Droplet-based microfluidics is an emerging interdisciplinary field with broad applications. On-demand droplet generation with precise size allows performing sensitive detection and quantitative analysis with high throughput and separate units, while the essential precondition is characterizing the volume of droplet accurately. Generally, fluorescence or bright-field imaging can be adopted to provide the length or width of the droplet from 2-D images, and then derive an idealized model to approximately estimate the droplet volume, which would always lead to steady-state errors with ignoring the irregular curvature of droplet surface in the depth direction. In this work, we propose the usage of real-time quantitative phase imaging (QPI) technique for the 3-D detection of flowing droplets to provide a precise measurement of droplet volume which can serve as feedback signal. Comparative experiments with a passive droplet generation system confirm that our proposed method can offer higher accuracy and monodispersity of on-demand droplet generation, together with enhanced stability and anti-interference to resist internal or external perturbations, which could promise a predictable outcome in many microfluidics-based systems.

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.

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.

Accurate quantitative phase imaging by differential phase contrast with partially coherent illumination: beyond weak object approximation

Quantitative phase imaging (QPI) by differential phase contrast (DPC) with partially coherent illumination provides speckle-free imaging and lateral resolution beyond the coherent diffraction limit, demonstrating great potential in biomedical imaging applications. Generally, DPC employs weak object approximation to linearize the phase-to-intensity image formation, simplifying the solution to the phase retrieval as a two-dimensional deconvolution with the corresponding phase transfer function. Despite its widespread adoption, weak object approximation still lacks a precise and clear definition, suggesting that the accuracy of the QPI results, especially for samples with large phase values, is yet to be verified. In this paper, we analyze the weak object approximation condition quantitatively and explicitly give its strict definition that is applicable to arbitrary samples and illumination apertures. Furthermore, an iterative deconvolution QPI technique based on pseudo-weak object approximation is proposed to overcome the difficulty of applying DPC to large-phase samples without additional data acquisition. Experiments with standard microlens arrays and MCF-7 cells demonstrated that the proposed method can effectively extend DPC beyond weak object approximation to high-precision three-dimensional morphological characterization of large-phase technical and biological samples.

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.

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.