Refractive Index Map Research Articles

State-of-the-art nondestructive high-speed raster scanning inspection for food safety and quality using terahertz refractive index mapping

Various methods are employed for food inspection; however, the persistent demand from both manufacturers and consumers remains due to the limitations of existing inspection technologies. This study introduces a novel technique for enhancing food safety and conducting large-area quality assessments using terahertz (THz) waves. This approach enables the classification of quality grades and the safe detection of soft matter, facilitated by high-speed mechanical raster scan imaging. Leveraging a two-dimensional complex refractive index map, we address the challenges posed by the uniformity in optical properties within food materials and foreign bodies, attributed to high water absorption and the presence of diverse compounds. Consequently, we anticipate improved foreign substance inspection and detection. These cutting-edge THz spectroscopic and imaging techniques, representing nondestructive inspection approaches, have the potential to elevate food safety and quality standards.

High-Density Polyethylene Custom Focusing Lenses for High-Resolution Transient Terahertz Biomedical Imaging Sensors.

Transient terahertz time-domain spectroscopy (THz-TDS) imaging has emerged as a novel non-ionizing and noninvasive biomedical imaging modality, designed for the detection and characterization of a variety of tissue malignancies due to their high signal-to-noise ratio and submillimeter resolution. We report our design of a pair of aspheric focusing lenses using a commercially available lens-design software that resulted in about 200 × 200-μm2 focal spot size corresponding to the 1-THz frequency. The lenses are made of high-density polyethylene (HDPE) obtained using a lathe fabrication and are integrated into a THz-TDS system that includes low-temperature GaAs photoconductive antennae as both a THz emitter and detector. The system is used to generate high-resolution, two-dimensional (2D) images of formalin-fixed, paraffin-embedded murine pancreas tissue blocks. The performance of these focusing lenses is compared to the older system based on a pair of short-focal-length, hemispherical polytetrafluoroethylene (TeflonTM) lenses and is characterized using THz-domain measurements, resulting in 2D maps of the tissue refractive index and absorption coefficient as imaging markers. For a quantitative evaluation of the lens effect on the image resolution, we formulated a lateral resolution parameter, R2080, defined as the distance required for a 20-80% transition of the imaging marker from the bare paraffin region to the tissue region in the same image frame. The R2080 parameter clearly demonstrates the advantage of the HDPE lenses over TeflonTM lenses. The lens-design approach presented here can be successfully implemented in other THz-TDS setups with known THz emitter and detector specifications.

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.

Real-time simultaneous refractive index and thickness mapping of sub-cellular biology at the diffraction limit

Mapping the cellular refractive index (RI) is a central task for research involving the composition of microorganisms and the development of models providing automated medical screenings with accuracy beyond 95%. These models require significantly enhancing the state-of-the-art RI mapping capabilities to provide large amounts of accurate RI data at high throughput. Here, we present a machine-learning-based technique that obtains a biological specimen’s real-time RI and thickness maps from a single image acquired with a conventional color camera. This technology leverages a suitably engineered nanostructured membrane that stretches a biological analyte over its surface and absorbs transmitted light, generating complex reflection spectra from each sample point. The technique does not need pre-existing sample knowledge. It achieves 10−4 RI sensitivity and sub-nanometer thickness resolution on diffraction-limited spatial areas. We illustrate practical application by performing sub-cellular segmentation of HCT-116 colorectal cancer cells, obtaining complete three-dimensional reconstruction of the cellular regions with a characteristic length of 30 μm. These results can facilitate the development of real-time label-free technologies for biomedical studies on microscopic multicellular dynamics.

Hyperspectral confocal imaging for high-throughput readout and analysis of bio-integrated microlasers.

Integrating micro- and nanolasers into live cells, tissue cultures and small animals is an emerging and rapidly evolving technique that offers noninvasive interrogation and labeling with unprecedented information density. The bright and distinct spectra of such lasers make this approach particularly attractive for high-throughput applications requiring single-cell specificity, such as multiplexed cell tracking and intracellular biosensing. The implementation of these applications requires high-resolution, high-speed spectral readout and advanced analysis routines, which leads to unique technical challenges. Here, we present a modular approach consisting of two separate procedures. The first procedure instructs users on how to efficiently integrate different types of lasers into living cells, and the second procedure presents a workflow for obtaining intracellular lasing spectra with high spectral resolution and up to 125-kHz readout rate and starts from the construction of a custom hyperspectral confocal microscope. We provide guidance on running hyperspectral imaging routines for various experimental designs and recommend specific workflows for processing the resulting large data sets along with an open-source Python library of functions covering the analysis pipeline. We illustrate three applications including the rapid, large-volume mapping of absolute refractive index by using polystyrene microbead lasers, the intracellular sensing of cardiac contractility with polystyrene microbead lasers and long-term cell tracking by using semiconductor nanodisk lasers. Our sample preparation and imaging procedures require 2 days, and setting up the hyperspectral confocal microscope for microlaser characterization requires <2 weeks to complete for users with limited experience in optical and software engineering.

Multi-harmonic structured illumination-based optical diffraction tomography

Imaging speed and spatial resolution are key factors in optical diffraction tomography (ODT), while they are mutually exclusive in 3D refractive index imaging. This paper presents a multi-harmonic structured illumination-based optical diffraction tomography (MHSI-ODT) to acquire 3D refractive index (RI) maps of transparent samples. MHSI-ODT utilizes a digital micromirror device (DMD) to generate structured illumination containing multiple harmonics. For each structured illumination orientation, four spherical spectral crowns are solved from five phase-shifted holograms, meaning that the acquisition of each spectral crown costs 1.25 raw images. Compared to conventional SI-ODT, which retrieves two spectral crowns from three phase-shifted raw images, MHSI-ODT enhances the imaging speed by 16.7% in 3D RI imaging. Meanwhile, MHSI-ODT exploits both the 1st-order and the 2nd-order harmonics; therefore, it has a better intensity utilization of structured illumination. We demonstrated the performance of MHSI-ODT by rendering the 3D RI distributions of 5 µm polystyrene (PS) microspheres and biological samples.

#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.

Continuous-wave terahertz in-line holographic diffraction tomography with the scattering fields reconstructed by a physics-enhanced deep neural network

Diffraction tomography is a promising, quantitative, and nondestructive three-dimensional (3D) imaging method that enables us to obtain the complex refractive index distribution of a sample. The acquisition of the scattered fields under the different illumination angles is a key issue, where the complex scattered fields need to be retrieved. Presently, in order to develop terahertz (THz) diffraction tomography, the advanced acquisition of the scattered fields is desired. In this paper, a THz in-line digital holographic diffraction tomography (THz-IDHDT) is proposed with an extremely compact optical configuration and implemented for the first time, to the best of our knowledge. A learning-based phase retrieval algorithm by combining the physical model and the convolution neural networks, named the physics-enhanced deep neural network (PhysenNet), is applied to reconstruct the THz in-line digital hologram, and obtain the complex amplitude distribution of the sample with high fidelity. The advantages of the PhysenNet are that there is no need for pretraining by using a large set of labeled data, and it can also work for thick samples. Experimentally with a continuous-wave THz laser, the PhysenNet is first demonstrated by using the thin samples and exhibits superiority in terms of imaging quality. More importantly, with regard to the thick samples, PhysenNet still works well, and can offer 2D complex scattered fields for diffraction tomography. Furthermore, the 3D refractive index maps of two types of foam sphere samples are successfully reconstructed by the proposed method. For a single foam sphere, the relative error of the average refractive index value is only 0.17%, compared to the commercial THz time-domain spectroscopy system. This demonstrates the feasibility and high accuracy of the THz-IDHDT, and the idea can be applied to other wavebands as well.

Synthetic Photoconductive Antenna Array for Terahertz 3-D Imaging and Spectroscopy

We propose a 3D imaging and spectroscopy approach in the terahertz (THz) domain based on the synthetic aperture (SA) imaging technique achieved using a time domain spectroscopy system (TDS). The proposed system enables the imaging and spectroscopy of an object in a stationary position in transmission configuration. We demonstrate the reconstruction of 3D amplitude images with a sub-millimeter resolution of a multi-material object from 0.6 to 1 THz using the back-projection algorithm. The reconstructed data is further used to characterize the optical properties of the substrates forming the object by calculating the refractive index and the absorption coefficient of its constituents in the reconstruction frequency band. We demonstrate the use of the SA not only for imaging, but also for material characterization. Consequently, we are able to reconstruct 2D high-resolution spectral maps of the real refractive index at different THz frequencies of the imaged object.

Mapping nanoscale topographic features in thick tissues with speckle diffraction tomography

Resolving three-dimensional morphological features in thick specimens remains a significant challenge for label-free imaging. We report a new speckle diffraction tomography (SDT) approach that can image thick biological specimens with ~500 nm lateral resolution and ~1 μm axial resolution in a reflection geometry. In SDT, multiple-scattering background is rejected through spatiotemporal gating provided by dynamic speckle-field interferometry, while depth-resolved refractive index maps are reconstructed by developing a comprehensive inverse-scattering model that also considers specimen-induced aberrations. Benefiting from the high-resolution and full-field quantitative imaging capabilities of SDT, we successfully imaged red blood cells and quantified their membrane fluctuations behind a turbid medium with a thickness of 2.8 scattering mean-free paths. Most importantly, we performed volumetric imaging of cornea inside an ex vivo rat eye and quantified its optical properties, including the mapping of nanoscale topographic features of Dua’s and Descemet’s membranes that had not been previously visualized.

Refractive index estimation in biological tissues by quantitative phase imaging

The refractive index (RI) of biological tissues represents a relevant optical property that correlates with intrinsic factors such as dry mass or water content, providing valuable information about tissue composition. First, we review available methods to measure the RI in biological tissues, making special emphasis on quantitative phase imaging (QPI) and its ability to obtain RI maps as well as tri-dimensional projections of transparent samples at diffraction-limited resolution. Second, we propose a simple, general QPI method to estimate the RI of complex materials without prior knowledge of sample thickness. Here we have tested several samples in various immersion media with different refractive indices (RIs) to use the lineal relation between the optical path difference (OPD) and the RI of the medium. Least squares regression operates to simultaneously determine the RI and the thickness with diffraction-limited resolution and nanometric transversal sensitivity. Our findings categorize a set of immersion media covering a good RI range, identify possible error sources and uncover the applicability of the QPI method, paving the way to its use on heterogeneous biological tissues.

Ultraviolet light scattering scanning flow cytometry in the characterization of submicron microparticles.

Ultraviolet lasers are commonly used in flow cytometry to excite fluorochrome molecules with subsequent measurement of the specific fluorescence of individual cells. In this study, the performance of the ultraviolet light scattering (UVLS) in the analysis of individual particles with flow cytometry has been demonstrated for the first time. The main advantage of the UVLS relates to the improvement of the analysis of submicron particles due to the strong dependence of the scattering efficiency on the wavelength of the incident light. In this work, submicron particles were analyzed using a scanning flow cytometer (SFC) that allows measurements of light scattering in an angle-resolved regime. The measured light-scattering profiles of individual particles were utilized in solution of the inverse light-scattering problem to retrieve the particle characteristics using a global optimization. The standard polystyrene microspheres were successfully characterized from the analysis of UVLS which provided the size and refractive index (RI) of individual beads. We believe that the main application of UVLS relates to the analysis of microparticles in a serum, in particular in the analysis of chylomicrons (CMs). We have demonstrated the performance of the UVLS SFC in the analysis of CMs of a donor. The RI versus size scatterplot of CMs was successfully retrieved from the analysis. The current set-up of the SFC has allowed us to characterize individual CMs starting from the size of 160 nm that provides determination of the CM concentration in a serum with flow cytometry. This feature of the UVLS should help with the analysis of lipid metabolism measuring RI and size map evolution after lipase action.

In-silico clearing approach for deep refractive index tomography by partial reconstruction and wave-backpropagation

Refractive index (RI) is considered to be a fundamental physical and biophysical parameter in biological imaging, as it governs light-matter interactions and light propagation while reflecting cellular properties. RI tomography enables volumetric visualization of RI distribution, allowing biologically relevant analysis of a sample. However, multiple scattering (MS) and sample-induced aberration (SIA) caused by the inhomogeneity in RI distribution of a thick sample make its visualization challenging. This paper proposes a deep RI tomographic approach to overcome MS and SIA and allow the enhanced reconstruction of thick samples compared to that enabled by conventional linear-model-based RI tomography. The proposed approach consists of partial RI reconstruction using multiple holograms acquired with angular diversity and their backpropagation using the reconstructed partial RI map, which unambiguously reconstructs the next partial volume. Repeating this operation efficiently reconstructs the entire RI tomogram while suppressing MS and SIA. We visualized a multicellular spheroid of diameter 140 µm within minutes of reconstruction, thereby demonstrating the enhanced deep visualization capability and computational efficiency of the proposed method compared to those of conventional RI tomography. Furthermore, we quantified the high-RI structures and morphological changes inside multicellular spheroids, indicating that the proposed method can retrieve biologically relevant information from the RI distribution. Benefitting from the excellent biological interpretability of RI distributions, the label-free deep visualization capability of the proposed method facilitates a noninvasive understanding of the architecture and time-course morphological changes of thick multicellular specimens.

Spectrometer-based wavelength interrogation SPR imaging via Hadamard transform.

We present spectrometer-based wavelength interrogation surface plasmon resonance imaging (SPRi) without mechanical scanning. A polarized broadband light source illuminates an object via a gold-coated prism; the reflected light is spatially modulated by a digital mirror device (DMD) and then measured with a spectrometer. Reflectance spectral images are reconstructed via the Hadamard transform (HT), and a refractive index (RI) map is visualized from the reflectance spectral images by analyzing the resonance peak shift of the spectrum at each image pixel. We demonstrate the feasibility of our method by evaluating the resolution, sensitivity, and dynamic detection range, experimentally obtained as ∼2.203 × 10-6 RI unit (RIU), ∼3,407 nm/RIU, and ∼0.1403 RIU, respectively. Furthermore, simulations are performed to validate the experimental results.

Squid Skin Cell-Inspired Refractive Index Mapping of Cells, Vesicles, and Nanostructures

The fascination with the optical properties of naturally occurring systems has been driven in part by nature's ability to produce a diverse palette of vibrant colors from a relatively small number of common structural motifs. Within this context, some cephalopod species have evolved skin cells called iridophores and leucophores whose constituent ultrastructures reflect light in different ways but are composed of the same high refractive index material─a protein called reflectin. Although such natural optical systems have attracted much research interest, measuring the refractive indices of biomaterial-based structures across multiple different environments and establishing theoretical frameworks for accurately describing the obtained refractive index values has proven challenging. Herein, we employ a synergistic combination of experimental and computational methodologies to systematically map the three-dimensional refractive index distributions of model self-assembled reflectin-based structures both in vivo and in vitro. When considered together, our findings may improve understanding of squid skin cell functionality, augment existing methods for characterizing protein-based optical materials, and expand the utility of emerging holotomographic microscopy techniques.

3D differential interference contrast microscopy using polarisation-sensitive tomographic diffraction microscopy.

Tomographic diffraction microscopy (TDM) is a generalisation of digital holographic microscopy (DHM), for which the illumination angle onto the sample is fully controlled, which has become a tool of choice for 3D, high-resolution imaging of unlabelled samples. TDM makes it possible to obtain the optical field in both amplitude and phase for each illumination angle. Proper information reallocation eventually allows for 3D reconstruction of the complex refractive index map. On the other hand, polarisation array sensors (PAS) paves new way for TDM, as vectorial information assessment about the investigated sample. In this contribution, we show an alternative use of this polarisation information based on the phase sensitive nature of TDM. Here, we demonstrated that TDM coupled with PAS can lead to a 3D differential interference contrast (DIC) microscope with almost no experimental configurationmodification.

Gradients of Orientation, Composition, and Hydration of Proteins for Efficient Light Collection by the Cornea of the Horseshoe Crab.

The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. The cornea of these eyes contains hundreds of inward projecting elongated cuticular cones and concentrate light onto proximal photoreceptor cells. Although this visual system has been extensively studied before, the precise mechanism allowing vision has remained controversial. Correlating high-resolution quantitative refractive index (RI) mapping and structural analysis, it is demonstrated how gradients of RI in the cornea stem from structural and compositional gradients in the cornea. In particular, these RI variations result from the chitin-protein fibers architecture, heterogeneity in protein composition, and bromine doping, as well as spatial variation in water content resulting from matrix cross-linking on the one hand and cuticle porosity on the other hand. Combining the realistic cornea structure and measured RI gradients with full-wave optical modeling and ray tracing, it is revealed that the light collection mechanism switches from refraction-based graded index (GRIN) optics at normal light incidence to combined GRIN and total internal reflection mechanism at high incident angles. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle.

Recovery of continuous 3D refractive index maps from discrete intensity-only measurements using neural fields

Intensity diffraction tomography (IDT) refers to a class of optical microscopy techniques for imaging the three-dimensional refractive index (RI) distribution of a sample from a set of two-dimensional intensity-only measurements. The reconstruction of artefact-free RI maps is a fundamental challenge in IDT due to the loss of phase information and the missing-cone problem. Neural fields has recently emerged as a new deep learning approach for learning continuous representations of physical fields. The technique uses a coordinate-based neural network to represent the field by mapping the spatial coordinates to the corresponding physical quantities, in our case the complex-valued refractive index values. We present Deep Continuous Artefact-free RI Field (DeCAF) as a neural-fields-based IDT method that can learn a high-quality continuous representation of a RI volume from its intensity-only and limited-angle measurements. The representation in DeCAF is learned directly from the measurements of the test sample by using the IDT forward model without any ground-truth RI maps. We qualitatively and quantitatively evaluate DeCAF on the simulated and experimental biological samples. Our results show that DeCAF can generate high-contrast and artefact-free RI maps and lead to an up to 2.1-fold reduction in the mean squared error over existing methods. Producing high-quality 3D refractive index maps from 2D intensity-only measurements is a long-standing objective in computational microscopy, with many applications involving the visualization of cellular and subcellular structures. A new method can reconstruct high-contrast and artefact-free images by employing the neural fields technique, which can learn a continuous 3D representation using a neural network that maps spatial coordinates to the refractive index values.

Developing a Reliable Holographic Flow Cyto-Tomography Apparatus by Optimizing the Experimental Layout and Computational Processing.

Digital Holographic Tomography (DHT) has recently been established as a means of retrieving the 3D refractive index mapping of single cells. To make DHT a viable system, it is necessary to develop a reliable and robust holographic apparatus in order that such technology can be utilized outside of specialized optics laboratories and operated in the in-flow modality. In this paper, we propose a quasi-common-path lateral-shearing holographic optical set-up to be used, for the first time, for DHT in a flow-cytometer modality. The proposed solution is able to withstand environmental vibrations that can severely affect the interference process. Furthermore, we have scaled down the system while ensuring that a full 360° rotation of the cells occurs in the field-of-view, in order to retrieve 3D phase-contrast tomograms of single cells flowing along a microfluidic channel. This was achieved by setting the camera sensor at 45° with respect to the microfluidic direction. Additional optimizations were made to the computational elements to ensure the reliable retrieval of 3D refractive index distributions by demonstrating an effective method of tomographic reconstruction, based on high-order total variation. The results were first demonstrated using realistic 3D numerical phantom cells to assess the performance of the proposed high-order total variation method in comparison with the gold-standard algorithm for tomographic reconstructions: namely, filtered back projection. Then, the proposed DHT system and the processing pipeline were experimentally validated for monocytes and mouse embryonic fibroblast NIH-3T3 cells lines. Moreover, the repeatability of these tomographic measurements was also investigated by recording the same cell multiple times and quantifying the ability to provide reliable and comparable tomographic reconstructions, as confirmed by a correlation coefficient greater than 95%. The reported results represent various steps forward in several key aspects of in-flow DHT, thus paving the way for its use in real-world applications.

On the hydrodynamic mutual interactions among cells for high-throughput microfluidic holographic cyto-tomography

In digital holography (DH) modality for lab on chip application the cells passing through the field of view (FOV) of the microscope can be detected and analyzed even if they are flowing at different depths. In fact, in-focus imaging of each cell can be easily retrieved thanks to the ability of DH to obtain numerical focus ex-post the recording process. An advantageous and preferred configuration for DH in flow-cytometry modality provides that the cells rotate along the microfluidic channel. This gives the unique chance to probing cells by light beams alongside many different directions while they cross the holographic FOV. Thus, it is possible to retrieve the 3D refractive index map of each flowing cell, i.e. a 3D phase-contrast tomogram. Although many cells can be analyzed in the same FOV, thus giving the possibility to increase the throughput of the system, until now no investigations have been made to establish how close the cells can be to avoid mutual disturbing effects on their rotation. Nevertheless, to estimate the maximum achievable throughput, it is indispensable to comprehend the effects of hydrodynamic interactions on adjacent cells in a tomographic flow cytometer. Here we show by means of an experimental and a numerical simulation of the fluid dynamics that hydrodynamic interactions have a quantitative effect on cells rotational behavior and neither on their mechanical deformation. However, in the considered scenario we demonstrate that the effect is negligible as it does not affect the possibility of recovering the tomograms. The reported results will allow to estimate which is the optimum density of cells to analyze by the holographic flow-cyto-tomograph thus opening the route for biomedical applications.