Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications

Inflammatory bowel diseases (IBD) are inflammatory disorders of the gastrointestinal tract characterized by a chronic relapsing disease course. As uncontrolled intestinal inflammation can result in severe disease complications, recent treatment targets of IBD evolved toward seeking the absence of mucosal and histological inflammation. However, this approach requires adequate histological evaluation of IBD disease activity. The diagnostic challenge of histological examination of intestinal inflammation is documented by the multitude of proposed histological scoring systems. In this context, we review quantitative phase imaging (QPI) techniques such as digital holographic microscopy (DHM) for characterizing intestinal inflammation. DHM determines optical path-length delays in a stain-free manner, thereby providing the tissue refractive index as a biophysical marker that directly correlates to tissue density. Recently, DHM has been successfully applied in cell biology, cancer cell research and infectious-induced cellular alterations. We summarized the capabilities of DHM and related QPI techniques to assess the severity of intestinal inflammation in experimental colitis as well as in colonic samples from human IBD patients. Moreover, we illustrate major advantages of DHM facilitated multimodal evaluation of epithelial wound healing processes as assessed by physical parameters like cell volume, density, thickness and dry mass in vitro. Furthermore, potential limitations of DHM and future utilities of QPI are discussed. In conclusion, DHM represents a promising, easy-to-use quantitative tool to provide accurate and objective assessment of intestinal inflammation and may pave the way towards automated label-free digital pathology and related in vitro cell culture analysis in future.

We report the characterization of all the relevant biomechanical properties of individual red blood cells with sickle cell disease using non-invasive quantitative phase imaging and spectroscopy techniques with a previously-validated RBC membrane model.

Biological cells are composed primarily of water; and as such are challenging to image without staining since the induced intensity modulation of transmitted or reflected light is typically insufficient to permit acceptable contrast for optical imaging. This issue may be resolved with the aid of exogenous contrast agents, but this often has a deleterious effect on the cell and precludes in vivo imaging. A unique approach to this problem is afforded by the phase contrast microscope in which optical-path differences in transmitted light is exploited as a contrast mechanism for qualitative imaging. In recent years however, several quantitative phase imaging techniques have been developed which allow for diffraction limited endogenous-contrast imaging with excellent temporal resolution. We hereby present a laser scanning technique for quantitative phase imaging which achieves sub-diffraction limited resolution at the expense of temporal resolution. This instrument is based on a stabilized fiber interfometer which is incorporated into a near-field scanning optical microscope (NSOM) for tri-modal imaging. Our latest results will focus on modifications made to this system to facilitate imaging in a liquid environment. A simple approach for achieving stable shear-force feedback operation in a liquid will be presented. Acquired high resolution images of white blood cells revealed detailed sub-cellular features. Images of fibroblast cells in air and in a liquid environment confirm the efficacy of the feedback operation in a liquid. Moreover, we demonstrate cell refractometry capability without the need for ad hoc modifications. These results clearly highlight the unique potential of this instrument for the study of living cells.

It is well known that the quantitative phase information which is vital in the biomedical study is hard to be directly obtained with bright-field microscopy under incoherent illumination. In addition, it is impossible to maintain the living sample in focus over long-term observation. Therefore, both the autofocusing and quantitative phase imaging techniques have to be solved in microscopy simultaneously. Here, we propose a lightweight deep learning-based framework, which is constructed by residual structure and is constrained by a novel loss function model, to realize both autofocusing and quantitative phase imaging. It outputs the corresponding in-focus amplitude and phase information at high speed (10fps) from a single-shot out-of-focus bright-field image. The training data were captured with a designed system under a hybrid incoherent and coherent illumination system. The experimental results verify that the focused and quantitative phase images of non-biological samples and biological samples can be reconstructed by using the framework. It provides a versatile quantitative technique for continuous monitoring of living cells in long-term and label-free imaging by using a traditional incoherent illumination microscopy system.

Quantitative phase imaging (QPI) technique is used to determine various biophysical parameters, such as refractive index, cell thickness, morphology, etc. On the other hand, fluorescence microscopy is used to acquire information regarding molecular specificity of the biological cells and tissues. Conventionally, a fully coherent light source such as laser is used in QPI technique to obtain the interference fringes with ease; however, its high coherence is also responsible for the generation of speckle and spurious fringes, which results in degraded image quality and affects the phase measurement results too. In this paper, we report a multi-modal system that can be effectively utilized to acquire time varied diverse information about the biological specimen with high spatial phase sensitivity. Herein, a single unit comprising of a fluorescence microscope and the Linnik based interferometer specially equipped with a partially spatially coherent light source illumination was developed. The integrated system is capable to procure molecular specificity and phase information of biological specimen, in a single shot, utilizing a single-chip color CCD camera. Here, we performed experiments on MG63 osteosarcoma cells, and the composite interferometric-fluorescence images were obtained and then digitally decomposed into red and green colors; and, the phase maps were reconstructed using the Fourier fringe analysis method. Furthermore, the cultured cells were monitored over a time-span to observe and investigate the time dependent morphological changes along with the quantification of cellular adhesion and spreading. Hence, the proposed system can be utilized to quantify time dependent changes in the cell’s morphology and in cell adhesion which can be an indicator for the detection of various range of diseases such as arthritis, cancer, osteoporosis and atherosclerosis.

Existing quantitative phase imaging (QPI) techniques are faced with an inherent trade-off between phase imaging fidelity and temporal resolution. Here, we propose a general algorithmic framework for QPI reconstruction that enables frame-rate-limited holographic imaging. It takes an inverse problem approach by formulating phase retrieval as a nonsmooth nonconvex optimization problem. Efficient solvers for the problem are derived whose algorithmic behaviors have been studied from both theoretical and experimental perspectives. The proposed framework is applicable to various existing holographic imaging configurations, and makes it possible to incorporate advanced image priors for quality enhancement.

White light diffraction phase microscopy with slightly off-axis blind two-step phase-shifting

This paper introduces a procedure aimed to quantitatively measure the optical properties of nanoparticles, namely the complex polarizability and the extinction, scattering, and absorption cross sections, simultaneously. The method is based on the processing of intensity and wavefront images of a light beam illuminating the nanoparticle of interest. Intensity and wavefront measurements are carried out using quadriwave lateral shearing interferometry, a quantitative phase imaging technique with high spatial resolution and sensitivity. The method does not require any preknowledge on the particle and involves a single interferogram image acquisition. The full determination of the actual optical properties of nanoparticles is of particular interest in plasmonics and nanophotonics for the active search and characterization of new materials, e.g., aimed to replace noble metals in future applications of nanoplasmonics with less-lossy or refractory materials.

We present quantitative optical measurements of individual human red blood cells from both diabetes patients and healthy blood donors using 3-D quantitative phase imaging technique in a noninvasive manner.

Interaction between intracellular compartments is based on complex vesicular transport processes. Fluorescence based techniques now reach single molecule resolution in living cells. To avoid fluorophore photobleaching limitations, quantitative phase imaging techniques are conventionally used to produce long-term high contrast cell images. We used an improved wave front sensing set-up called QWLSI-HD, which resolution is high enough to reach nanometric resolution for vesicle localization. We also added specificity to quantitative phase imaging by training artificial intelligence algorithms to recognize intracellular vesicles. We compared performances between the algorithms and also between fixed and living samples.

We propose a quantitative phase imaging technique with single-path phase-shifting digital holography using a light-emitting diode (LED). A reference wave is generated from an object wave in the Fourier plane using a single-path interferometer, based on self-reference digital holography. The object wave interferes with the reference wave, and the quantitative phase information of the object wave is recorded as a digital hologram. Quantitative phase images of objects are obtained by applying a phase-shifting interferometry technique. All the light diffracted from the objects can be utilized to generate a digital hologram. Its validity is experimentally demonstrated by constructing an optical system with a wide-field optical microscope.

Revealing the structural and functional organization of biological samples at submicrometer scales requires high-resolution imaging techniques that provide real-time, quantitative information. However, resolving and analyzing unlabeled biological structures at the subcellular level from a single-shot image remain challenging. We introduce a novel incoherent quantitative phase imaging technique based on lateral shearing digital holographic microscopy, with shearing distances exceeding the coherence area of the employed illumination—a regime not previously achieved and exploited. This strategy consequently ensures artifact-free, high-accuracy, and high-resolution quantitative phase reconstructions. This regime extends single-shot lateral shearing digital holographic microscopy toward incoherent illumination, thus increasing the space–time bandwidth product of the method. The practical common-path geometry also provides enhanced vibration resistance, which is essential for consistent time-lapse measurements. We verify the scalability and effectiveness of the method by investigating samples via multiple condenser–objective pairs, providing a wide range of lateral resolutions and fields of view, thereby ensuring its applicability for various microscope settings. We experimentally verify long-term phase stability and unprecedented phase-reconstruction accuracy. Finally, we use our method to investigate biological samples, including cheek cells, diatoms, and yeast cells, highlighting its potential for dynamic label-free analysis at the organelle level. These results establish our approach as a powerful tool for quantitative phase imaging, enabling high-precision investigations across a wide range of scientific applications.

Hybrid Random Walk-Linear Discriminant Analysis Method for Unwrapping Quantitative Phase Images of Biological Samples

Conventional optical microscopy is one of the most commonly used method in biological imaging studies. However, intensity-based imaging techniques exhibit poor imaging contrasts when biological samples are considered. Several methods, like phase contrast microscopy or darkfield microscopy, have been proposed to overcome these limitations. A better qualitative information can then be extracted at the cost of experimental devices multiplicity. On the other hand, quantitative phase imaging techniques like Tomographic Diffraction Microscopy (TDM) make it possible to extract full 3D information about the light field emerging from the investigated sample. In this article, we propose to take benefits of TDM capabilities to simulate the behavior of conventional optical microscope, making TDM and universal microscopy imaging platform.

Optical diffraction tomography (ODT) is a three-dimensional (3D) quantitative phase imaging technique, which enables the reconstruction of the 3D refractive index (RI) distribution of a transparent sample. Due to its fast, non-invasive, and quantitative imaging capability, ODT has emerged as a powerful tool for various applications. However, the spatial resolution of ODT has only been quantified along the lateral and axial directions for limited conditions; it has not been investigated for arbitrary-oblique directions. In this paper, we systematically quantify the 3D spatial resolution of ODT by exploiting the spatial bandwidth of the reconstructed scattering potential. The 3D spatial resolution is calculated for various types of systems, including the illumination-scanning, sample-rotation, and hybrid scanning-rotation methods. In particular, using the calculated 3D spatial resolution, we provide the spatial resolution as well as the arbitrary sliced angle. Furthermore, to validate the present method, the point spread function of an ODT system is experimentally obtained using the deconvolution of a 3D RI distribution of a microsphere and is compared with the calculated resolution.