Three-dimensional Refractive Index Research Articles

In silico full-angle high-dynamic range scattering of microscopic objects exploiting holotomography.

Accurate optical characterization of microscopic objects is crucial in academic research, product development, and clinical diagnosis. We present a method for obtaining full and high-dynamic range, angle-resolved light scattering attributes of microparticles, enabling distinction of variations in both overall morphology and detailed internal structures. This method overcomes previous limitations in observable scattering angles and dynamic range of signals through computationally assisted three-dimensional holotomography. This advancement is significant for particles spanning tens of wavelengths, such as human erythrocytes, which have historically posed measurement challenges due to faint side-scattering signals indicative of their complex interiors. Our technique addresses three key challenges in optical side-scattering analysis: limited observational angular range, reliance on simplified computational models, and low signal-to-noise ratios in both experimental and computational evaluations. We incorporate three-dimensional tomographic complex refractive index data from Fourier-transform light scattering into a tailored finite-difference time-domain simulation space. This approach facilitates precise near-to-far-field transformations. The process yields complete full-angle scattering phase functions, crucial for particles like Plasmodium falciparum-parasitized erythrocytes, predominantly involved in forward scattering. The resultant scattering data exhibit an extreme dynamic range exceeding 100 dB at various incident angles of a He-Ne laser. These findings have the potential to develop point-of-care, cost-effective, and rapid malaria diagnostic tools, inspiring further clinical and research applications in microparticle scattering.

Bidirectional in-silico clearing approach for deep refractive-index tomography using a sparsely sampled transmission matrix.

Optical diffraction tomography (ODT) enables the label-free volumetric imaging of biological specimens by mapping their three-dimensional refractive index (RI) distribution. However, the depth of imaging achievable is restricted due to spatially inhomogeneous RI distributions that induce multiple scattering. In this study, we introduce a novel ODT technique named bidirectional in-silico clearing RI tomography. This method incorporates both forward and reversed in-silico clearing. For the reversed in-silico clearing, we have integrated an ODT reconstruction framework with a transmission matrix approach, which enables RI reconstruction and wave backpropagation from the illumination side without necessitating modifications to the conventional ODT setup. Furthermore, the framework employs a sparsely sampled transmission matrix, significantly reducing the requisite number of measurements and computational expenses. Employing this proposed technique, we successfully imaged a spheroid with a thickness of 263 µm, corresponding to 11.4 scattering mean free paths. This method was successfully applied to various biological specimens, including liver and colon spheroids, demonstrating consistent imaging performance across samples with varied morphologies.

Single-exposure multi-wavelength optical diffraction tomography based on space-angle dual multiplexing holography.

We present a space-angle dual multiplexing holographic recording system for realizing single-exposure multi-wavelength optical diffraction tomographic (ODT) imaging. This system is achieved by combining the principle of single-exposure multi-wavelength holographic imaging technique based on angle-division multiplexing with the principle of single-exposure ODT imaging technique based on microlens array multi-angle illuminations and space-division multiplexing. Compared with the existing multi-wavelength ODT imaging methods, it enables the holographic recording of all the diffraction tomography information of a measured specimen at multiple illumination wavelengths in a single camera exposure without any scan mechanism. Using our proposed data processing method, the multi-wavelength three-dimensional (3D) refractive index tomograms of a specimen can be eventually reconstructed from single recorded multiplexing hologram. Experimental results of a static polystyrene bead and a living C. elegans worm demonstrate the feasibility of this system.

Label-Free Three-Dimensional Morphological Characterization of Cell Death Using Holographic Tomography.

This study presents a novel label-free approach for characterizing cell death states, eliminating the need for complex molecular labeling that may yield artificial or ambiguous results due to technical limitations in microscope resolution. The proposed holographic tomography technique offers a label-free avenue for capturing precise three-dimensional (3D) refractive index morphologies of cells and directly analyzing cellular parameters like area, height, volume, and nucleus/cytoplasm ratio within the 3D cellular model. We showcase holographic tomography results illustrating various cell death types and elucidate distinctive refractive index correlations with specific cell morphologies complemented by biochemical assays to verify cell death states. These findings hold promise for advancing in situ single cell state identification and diagnosis applications.

Alternating projection combined with fast gradient projection (FGP-AP) method for intensity-only measurement optical diffraction tomography in LED array microscopy.

Optical diffraction tomography (ODT) is a powerful label-free measurement tool that can quantitatively image the three-dimensional (3D) refractive index (RI) distribution of samples. However, the inherent "missing cone problem," limited illumination angles, and dependence on intensity-only measurements in a simplified imaging setup can all lead to insufficient information mapping in the Fourier domain, affecting 3D reconstruction results. In this paper, we propose the alternating projection combined with the fast gradient projection (FGP-AP) method to compensate for the above problem, which effectively reconstructs the 3D RI distribution of samples using intensity-only images captured from LED array microscopy. The FGP-AP method employs the alternating projection (AP) algorithm for gradient descent and the fast gradient projection (FGP) algorithm for regularization constraints. This approach is equivalent to incorporating prior knowledge of sample non-negativity and smoothness into the 3D reconstruction process. Simulations demonstrate that the FGP-AP method improves reconstruction quality compared to the original AP method, particularly in the presence of noise. Experimental results, obtained from mouse kidney cells and label-free blood cells, further affirm the superior 3D imaging efficacy of the FGP-AP method.

Numerical study of the effects of hydrodynamic interactions among cells for microfluidic holographic cyto-tomography

When cells in a suspension flow through a microfluidic channel and rotate within the field of view (FOV) of a holographic microscope, they become accessible to a light beam from various angles. This allows the retrieval of a three-dimensional refractive index map for each flowing cell, essentially a 3D phase-contrast tomogram. Understanding the effects of hydrodynamic interactions among cells on their rotational behaviour during flow is crucial for designing microfluidic devices for holographic imaging. In this study, we employ direct numerical simulations to investigate the dynamics of cell clusters suspended in a Newtonian liquid under pressure-driven flow within a microfluidic channel, with the aim of clarifying the influence of hydrodynamic interactions on cell rotation.

Spatial Fluctuations of Optical Turbulence Strength in a Laboratory Turbulence Simulator

Controlled turbulence simulators in the laboratory have been extensively employed to investigate turbulence effects on light propagation in the atmosphere, driven by some advanced optical engineering such as remote sensing, energy-delivery systems, and free-space optical communication systems. Many studies have achieved rich results on the optical turbulence intensity, scintillation index, and power spectral density characteristics of the light propagation path in the center of a turbulence simulator, but a comprehensive analysis of the optical turbulence characteristics for different spatial locations is still lacking. We simulate turbulence with air as the medium in a classical convective Rayleigh–Bénard turbulence simulator through high-resolution computational fluid dynamics methods, the three-dimensional refractive index distribution is obtained, and the optical properties are analyzed comprehensively. It is found that the hot and cold plumes and the large-scale circulation strongly influence the inhomogeneity of Cn2 in the turbulence tank, making it weak in the middle and strong near the boundary. The refractive index power spectral density at different heights is centrally symmetric, with the slope gradually deviating from the −5/3 scaling power with increasing distance from the central region. Under the log-log plot, the variation of the refractive index variance with height exhibits a three-segmented feature, showing in order: a stable region, a logarithmic profile, and a power-law profile, in the region close to the boundary. These results will contribute to the construction of a suitable turbulence simulator for optical engineering applications.

Reconstruction refinement of hybrid background-oriented schlieren tomography

This paper introduces a hybrid method that leverages the advantages of both the window-based separate algorithm and the regularization-based unifying algorithm to refine the three-dimensional refractive index field measured by the background-oriented schlieren (BOS) tomography. Two image-warping approaches, under paraxial and non-paraxial ray-tracing assumption, are developed as a connecting link between coarse and refined reconstruction. An orthogonal test is conducted to examine the influence of various factors on the reconstruction fields in the hybrid approach, providing insights into the impact of factors such as the ray-tracing assumption, initial value for refractive index iteration, and image displacement algorithm. Various synthetic and real BOS cases demonstrate that our hybrid method reconstructs a more detailed and robust field, outperforming conventional BOS reconstruction methods in scenarios with small or large density gradients, as well as laminar or turbulent structures.

A multi-core fiber coupler without a central core

An efficient method for fabricating multi-core fiber couplers based on the thermal diffusion technique is proposed to realize the connection of single-mode fibers to multi-core fibers without a central core. The fabrication efficiency of the multi-core fiber coupler is improved by fiber-loaded hydrogen. The simulation results show that the single-mode fiber and the multi-core fiber can be coupled stably and efficiently by using the designed double-clad fiber to connect the single-mode fiber and the multi-core fiber. Double-clad fiber and multi-core fiber were loaded with hydrogen to enhance the dopant diffusion rate. Three-dimensional refractive index measurements of the multi-core fiber coupler show that the dopant diffusion rate and the diffusion zone length of the hydrogen-loaded fiber are significantly increased compared to the original fiber. These enhancements contributed to the shorter fabrication time of multi-core fiber couplers. The multi-core fiber couplers fabricated by thermal diffusion technology have the advantages of high coupling efficiency, high integration, simple manufacture, and high mechanical strength. Enhancing the diffusion rate of optical fibers by using the hydrogen loading method will increase the application potential of multi-core optical fiber couplers prepared by thermal diffusion technology, and accelerate the translation of multi-core fibers toward optical communication and optical fiber sensing.

Quantitative real-time phase microscopy for extended depth-of-field imaging based on the 3D single-shot differential phase contrast (ssDPC) imaging method.

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.

Three-dimensional refractive index microscopy based on the multi-layer propagation model with obliquity factor correction

Multi-layer propagation model-based intensity diffraction tomography (IDT) is a three-dimensional (3D) microscopy imaging technique to reveal the structural information of optically thick or multiple scattering samples. However, the performance of existing multi-layer propagation models deteriorates with increasing illumination angle, which can prevent good 3D refractive index (RI) imaging. To overcome this limitation, we incorporate the obliquity factor (OF) into the multi-layer propagation model. Numerical simulation validates that the forward propagation and the RI reconstruction both can be considerably improved. Then, the 3D RI reconstruction performance is quantitatively and qualitatively validated by optical experiments on polystyrene microspheres and peony pollen grains, respectively. In addition, excellent optical sectioning and high spatial resolution are experimentally demonstrated on various unlabeled fixed and living samples, including an Arabidopsis root, HeLa and HCT-116 cells. Time-lapse 3D imaging of living samples is performed for the first time for multi-layer propagation model-based IDT, demonstrating its potential application to some biological and medical studies.

Enhanced functionalities of immune cells separated by a microfluidic lattice: assessment based on holotomography.

The isolation of white blood cells (WBCs) from whole blood constitutes a pivotal process for immunological studies, diagnosis of hematologic disorders, and the facilitation of immunotherapy. Despite the ubiquity of density gradient centrifugation in WBC isolation, its influence on WBC functionality remains inadequately understood. This research employs holotomography to explore the effects of two distinct WBC separation techniques, namely conventional centrifugation and microfluidic separation, on the functionality of the isolated cells. We utilize three-dimensional refractive index distribution and time-lapse dynamics to analyze individual WBCs in-depth, focusing on their morphology, motility, and phagocytic capabilities. Our observations highlight that centrifugal processes negatively impact WBC motility and phagocytic capacity, whereas microfluidic separation yields a more favorable outcome in preserving WBC functionality. These findings emphasize the potential of microfluidic separation techniques as a viable alternative to traditional centrifugation for WBC isolation, potentially enabling more precise analyses in immunology research and improving the accuracy of hematologic disorder diagnoses.

Refractive index tomography with a physics-based optical neural network.

The non-interference three-dimensional refractive index (RI) tomography has attracted extensive attention in the life science field for its simple system implementation and robust imaging performance. However, the complexity inherent in the physical propagation process poses significant challenges when the sample under study deviates from the weak scattering approximation. Such conditions complicate the task of achieving global optimization with conventional algorithms, rendering the reconstruction process both time-consuming and potentially ineffective. To address such limitations, this paper proposes an untrained multi-slice neural network (MSNN) with an optical structure, in which each layer has a clear corresponding physical meaning according to the beam propagation model. The network does not require pre-training and performs good generalization and can be recovered through the optimization of a set of intensity images. Concurrently, MSNN can calibrate the intensity of different illumination by learnable parameters, and the multiple backscattering effects have also been taken into consideration by integrating a "scattering attenuation layer" between adjacent "RI" layers in the MSNN. Both simulations and experiments have been conducted carefully to demonstrate the effectiveness and feasibility of the proposed method. Experimental results reveal that MSNN can enhance clarity with increased efficiency in RI tomography. The implementation of MSNN introduces a novel paradigm for RI tomography.

A Coaxial Dual-Waveguide Fiber Coupler

We demonstrate a method to fabricate an in-fiber integrated multi-waveguide fiber coupler based on thermal diffusion technology to achieve stable and efficient coupling of a single-mode fiber to a multi-waveguide fiber. Simulation results show that the efficient coupling of a single-mode fiber and a multi-waveguide fiber can be realized by introducing double-clad fiber into single-mode fiber and multi-waveguide fiber. A coaxial dual-waveguide fiber coupler was fabricated based on thermal diffusion technology. The measurement results of the three-dimensional refractive index distribution of the coaxial dual-waveguide fiber coupler verified the feasibility of the thermal diffusion technology to fabricate the multi-waveguide fiber coupler. The insertion loss of the thermally diffused coaxial dual-waveguide fiber coupler was tested, and its performance was better than that of the fusion tapered coupler. The multi-waveguide fiber coupler fabricated by thermal diffusion technology allows for an all-fiber setup with coupling efficiency as high as 98%, achieves a relatively stable fundamental mode excitation in multi-waveguide fiber, requires minimal maintenance, and will accelerate the translation toward particle trapping, spectral detection, and optical communication.

LED array microscopy system correction method with comprehensive error parameters optimized by phase smoothing criterion.

LED array microscopy is a novel computational imaging technique that can achieve two-dimensional (2D) phase imaging and three-dimensional (3D) refractive index imaging with both high resolution and a large field of view. Although its experimental setup is simple, the errors caused by LED array position and light source central wavelength obviously decrease the quality of reconstructed results. To solve this problem, comprehensive error parameters optimized by the phase smoothing criterion are put forward in this paper. The central wavelength error and 3D misalignment model with six freedom degree errors of LED array are considered as the comprehensive error parameters when the spatial positional and optical features of arbitrarily placed LED array are unknown. Phase smoothing criterion is also introduced to the cost function for optimizing comprehensive error parameters to improve the convergence results. Compared with current system correction methods, the simulation and experimental results show that the proposed method in this paper has the best reconstruction accuracy, which can be well applied to an LED array microscope system with unknown positional and optical features of the LED array.

Three-dimensional refractive index estimation based on deep-inverse non-interferometric optical diffraction tomography (ODT-Deep).

Optical diffraction tomography (ODT) solves an inverse scattering problem to obtain label-free, 3D refractive index (RI) estimation of biological specimens. This work demonstrates 3D RI retrieval methods suitable for partially-coherent ODT systems supported by intensity-only measurements consisting of axial and angular illumination scanning. This framework allows for access to 3D quantitative RI contrast using a simplified non-interferometric technique. We consider a traditional iterative tomographic solver based on a multiple in-plane representation of the optical scattering process and gradient descent optimization adapted for focus-scanning systems, as well as an approach that relies solely on 3D convolutional neural networks (CNNs) to invert the scattering process. The approaches are validated using simulations of the 3D scattering potential for weak phase 3D biological samples.

Quasi-Isotropic High-Resolution Fourier Ptychographic Diffraction Tomography with Opposite Illuminations

Optical diffraction tomography (ODT) is a powerful tool for the study of unlabeled biological cells thanks to its unique capability of measuring the three-dimensional (3D) refractive index (RI) distribution of samples quantitatively and noninvasively. In conventional transmission ODT, however, certain spatial frequency components along the optical axis cannot be measured due to the limited angular coverage of the incident beam, resulting in a poor axial resolution several times worse than the lateral one. In this Letter we propose a new type of ODT method, termed opposite illumination Fourier ptychographic diffraction tomography (OI-FPDT), which produces almost isotropic resolution by combining transmissive angle-scanning and reflective wavelength-scanning. Without resorting to interferometric detection, OI-FPDT requires an intensity-only measurement, and the forward and backward scattered intensity images are synthesized in the Fourier space to recover the 3D RI distribution of samples based on an iterative ptychographic reconstruction algorithm. To the best of our knowledge, this is the first time that near-isotropic resolution (∼ 274 nm) of ODT result is obtained in a non-interferometric and sample motion-free manner. Results of simulated cell phantom, tailor-made fiberglass, and onion epidermal cell samples confirm the validity of the proposed method.

Analysis of Acousto-Optic Phenomenon in SAW Acoustofluidic Chip and Its Application in Light Refocusing.

This paper describes and analyzes a common acousto-optic phenomenon in surface acoustic wave (SAW) microfluidic chips and accomplishes some imaging experiments based on these analyses. This phenomenon in acoustofluidic chips includes the appearance of bright and dark stripes and image distortion. This article analyzes the three-dimensional acoustic pressure field and refractive index field distribution induced by focused acoustic fields and completes an analysis of the light path in an uneven refractive index medium. Based on the analysis of microfluidic devices, a SAW device based on a solid medium is further proposed. This MEMS SAW device can refocus the light beam and adjust the sharpness of the micrograph. The focal length can be controlled by changing the voltage. Moreover, the chip is also proven to be capable of forming a refractive index field in scattering media, such as tissue phantom and pig subcutaneous fat layer. This chip has the potential to be used as a planar microscale optical component that is easy to integrate and further optimize and provides a new concept about tunable imaging devices that can be attached directly to the skin or tissue.

Label-Free Digital Holotomography Reveals Ibuprofen-Induced Morphological Changes to Red Blood Cells.

Understanding the dose-dependent effect of over-the-counter drugs on red blood cells (RBCs) is crucial for hematology and digital pathology. Yet, it is challenging to continuously record the real-time, drug-induced shape changes of RBCs in a label-free manner. Here, we demonstrate digital holotomography (DHTM)-enabled real-time, label-free concentration-dependent and time-dependent monitoring of ibuprofen on RBCs from a healthy donor. The RBCs are segmented based on three-dimensional (3D) and four-dimensional (4D) refractive index tomograms, and their morphological and chemical parameters are retrieved with their shapes classified using machine learning. We directly observed the formation and motion of spicules on the RBC membrane when aqueous solutions of ibuprofen were drop-cast on wet blood, creating rough-membraned echinocyte forms. At low concentrations of 0.25-0.50 mM, the ibuprofen-induced morphological change was transient, but at high concentrations (1-3 mM) the spiculated RBC remained over a period of up to 1.5 h. Molecular simulations confirmed that aggregates of ibuprofen molecules at high concentrations significantly disrupted the RBC membrane structural integrity and lipid order but produced negligible effect at low ibuprofen concentrations. Control experiments on the effect of urea, hydrogen peroxide, and aqueous solutions on RBCs showed zero spicule formation. Our work clarifies the dose-dependent chemical effects on RBCs using label-free microscopes that can be deployed for the rapid detection of overdosage of over-the-counter and prescribed drugs.

Parallel synthetic aperture transport-of-intensity diffraction tomography with annular illumination.

Transport-of-intensity diffraction tomography (TIDT) is a recently developed label-free computational microscopy technique that retrieves high-resolution three-dimensional (3D) refractive index (RI) distribution of biological specimens from 3D intensity-only measurements. However, the non-interferometric synthetic aperture in TIDT is generally achieved sequentially through the acquisition of a large number of through-focus intensity stacks captured at different illumination angles, resulting in a very cumbersome and redundant data acquisition process. To this end, we present a parallel implementation of a synthetic aperture in TIDT (PSA-TIDT) with annular illumination. We found that the matched annular illumination provides a mirror-symmetric 3D optical transfer function, indicating the analyticity in the upper half-plane of the complex phase function, which allows for recovery of the 3D RI from a single intensity stack. We experimentally validated PSA-TIDT by conducting high-resolution tomographic imaging of various unlabeled biological samples, including human breast cancer cell lines (MCF-7), human hepatocyte carcinoma cell lines (HepG2), Henrietta Lacks (HeLa) cells, and red blood cells (RBCs).