3D Refractive Index Research Articles

Tomographic background oriented schlieren using plenoptic cameras

The development of a tomographic background oriented schlieren implementation system utilizing up to four plenoptic cameras is presented. A systematic set of experiments was performed using a pair of solid dimethylpolysiloxan cylinders immersed in a nearly refractive index matched gylcerol/water solution to represent discrete flow features with known sizes, shapes, separation distances, and orientation. A study was conducted to assess the influence of these features on the accuracy of 3D reconstructions of the refractive index field. It was determined that the limited angular information collected by a single plenoptic camera is insufficient for single-camera 3D reconstructions. In multi-camera configurations, the additional views collected by a plenoptic camera were shown to improve the overall reconstruction accuracy compared to an equivalent single view per camera reconstruction, potentially reducing the number of overall cameras needed to achieve a desired accuracy. For the imaging of two cylinders, three or more cameras are generally needed to avoid significant ghosting artifacts in the reconstruction. Quantitative results are presented that show that: (1) two separate cylinders will be individually resolved as long as measurements from one camera are able to observe separation between the cylinders; (2) the error in the reconstructed 3D refractive index field increases as the size of the feature decreases; and (3) the use of volumetric masking within the reconstruction algorithm is critical in order to improve the accuracy of the solution.

Towards non-blind optical tweezing by finding 3D refractive index changes through off-focus interferometric tracking

In modern 3D microscopy, holding and orienting arbitrary biological objects with optical forces instead of using coverslips and gel cylinders is still a vision. Although optical trapping forces are strong enough and related photodamage is acceptable, the precise (re-) orientation of large specimen with multiple optical traps is difficult, since they grab blindly at the object and often slip off. Here, we present an approach to localize and track regions with increased refractive index using several holographic optical traps with a single camera in an off-focus position. We estimate the 3D grabbing positions around several trapping foci in parallel through analysis of the beam deformations, which are continuously measured by defocused camera images of cellular structures inside cell clusters. Although non-blind optical trapping is still a vision, this is an important step towards fully computer-controlled orientation and feature-optimized laser scanning of sub-mm sized biological specimen for future 3D light microscopy.

Dehydration of plant cells shoves nuclei rotation allowing for 3D phase-contrast tomography

Single-cell phase-contrast tomography promises to become decisive for studying 3D intracellular structures in biology. It involves probing cells with light at wide angles, which unfortunately requires complex systems. Here we show an intriguing concept based on an inherent natural process for plants biology, i.e., dehydration, allowing us to easily obtain 3D-tomography of onion-epidermal cells’ nuclei. In fact, the loss of water reduces the turgor pressure and we recognize it induces significant rotation of cells’ nuclei. Thanks to the holographic focusing flexibility and an ad-hoc angles’ tracking algorithm, we combine different phase-contrast views of the nuclei to retrieve their 3D refractive index distribution. Nucleolus identification capability and a strategy for measuring morphology, dry mass, biovolume, and refractive index statistics are reported and discussed. This new concept could revolutionize the investigation in plant biology by enabling dynamic 3D quantitative and label-free analysis at sub-nuclear level using a conventional holographic setup.

GRINCU lens with conicoid iso-indicial surfaces: application for modeling the crystalline lens.

We introduce a new type of lens with two gradients of refractive index (GRIN) and of curvature (GRCU) of iso-indicial surfaces, i.e., GRINCU. The inner structure of the lens resembles that of an onion. Each layer is a meniscus lens with infinitesimal thickness, which coincides with an iso-indicial surface characterized by a conicoid shape and a constant refractive index. The internal distribution automatically adapts to the external geometry. Here, we consider the simplest case of a constant gradient of the curvature radius -G, which indicates a linear decrease as we move along the optical axis. The formulation of this type of lens is presented, including its generalization to nonrotationally symmetric conicoid surfaces. The formulation is then applied to model the crystalline lens; the code corresponding to the numerical computation of the 3D refractive index distribution as well as its gradient is provided as a supplementary file. Finally, we confirmed a refractive power increase of nearly 14% when G changes from 0 to 3.

Metformin protects against diclofenac-induced toxicity in primary rat hepatocytes by preserving mitochondrial integrity via a pathway involving EPAC

Background and purposeIt has been shown that the antidiabetic drug metformin protects hepatocytes against toxicity by various stressors. Chronic or excessive consumption of diclofenac (DF) - a pain-relieving drug, leads to drug-induced liver injury via a mechanism involving mitochondrial damage and ultimately apoptotic death of hepatocytes. However, whether metformin protects against DF-induced toxicity is unknown. Recently, it was also shown that cAMP elevation is protective against DF-induced apoptotic death in hepatocytes, a protective effect primarily involving the downstream cAMP effector EPAC and preservation of mitochondrial function. This study therefore aimed at investigating whether metformin protects against DF-induced toxicity via cAMP-EPACs. Experimental approachPrimary rat hepatocytes were exposed to 400 µmol/L DF. CE3F4 or ESI-O5 were used as EPAC-1 or 2 inhibitors respectively. Apoptosis was measured by caspase-3 activity and necrosis by Sytox green staining. Seahorse X96 assay was used to determine mitochondrial function. Mitochondrial reactive oxygen species (ROS) production was measured using MitoSox, mitochondrial MnSOD expression was determined by immunostaining and mitochondrial morphology (fusion and fission ratio) by 3D refractive index imaging. Key resultsMetformin (1 mmol/L) was protective against DF-induced apoptosis in hepatocytes. This protective effect was EPAC-dependent (mainly EPAC-2). Metformin restored mitochondrial morphology in an EPAC-independent manner. DF-induced mitochondrial dysfunction which was demonstrated by decreased oxygen consumption rate, an increased ROS production and a reduced MnSOD level, were all reversed by metformin in an EPAC-dependent manner. Conclusion and implicationsMetformin protects hepatocytes against DF-induced toxicity via cAMP-dependent EPAC-2.

Label-Free Quantitative Phase Imaging Reveals Spatial Heterogeneity of Extracellular Vesicles in Select Colon Disorders

Three-dimensional (3D) imaging and quantitative analysis of extracellular vesicles (EVs) remain largely unexplored, mainly because of limitations in detection techniques. In this study, EVs from patients diagnosed with colorectal cancer (CRC) and ulcerative colitis were examined. To investigate the spatial heterogeneity and 3D refractive index (RI) distribution of single EVs, a label-free digital holographic tomography technique was used at a submicrometer spatial resolution. The presented image-processing algorithms were used in quantitative analysis with digital staining and 3D visualization, the determination of the EV size distribution and extraction of fractions with different RIs. Reconstructed 3D RI distributions revealed variations in the spatial heterogeneity of EVs related to tissue specificity, such as CRC, normal colonic mucosa, and ulcerative colitis, as well as the isolation procedures used. The RI values of EVs isolated from solid tissues of frozen CRC samples were also dependent on the tumor grade and cancer cell proliferation. The simultaneous examination of cell culture models confirmed the association of the RI of EVs with the tumor grade. 3D-RI data analysis generates new perspectives with the optical, contact-free, label-free examination of the individual EVs. Depending on the specific tissue and isolation method, EVs exhibit significant spatial heterogeneity. The optical parameters of single EVs enabled their classification into two unique subgroups with different RI values.

Reconstruction enhancement via projection screening in holographic tomography

This paper presents an algorithm for automatic detection of erroneous amplitude and phase components of a sample’s optical field, acquired by a holographic tomograph with a limited angle of projection. By applying image processing methods and statistical analysis to find and remove unfit projections, the quality of tomographic reconstruction of a 3D refractive index distribution of an object is greatly improved. The proposed methods can find their application in preprocessing of data in holographic tomography. Full Text: PDF ReferencesA. Kuś, W. Krauze, P. L. Makowski, and M. Kujawińska, "Holographic tomography: hardware and software solutions for 3D quantitative biomedical imaging (Invited paper)", ETRI Journal, 41, 1 (2019). CrossRef V. Balasubramani et al., "Phase unwrapping in ICF target interferometric measurement via deep learning", Appl. Opt., 60, 10 (2021). CrossRef Y. Park, C. Depeursinge, and G. Popescu, "Quantitative phase imaging in biomedicine", Nature Photonics, 12, 10 (2018). CrossRef W. Krauze, P. Makowski, M. Kujawińska, and A. Kuś, "Generalized total variation iterative constraint strategy in limited angle optical diffraction tomography", Opt. Express, 24, 5 (2016). CrossRef D. Ryu et al., "A non-calorimetric approach for investigating the moisture-induced ageing of a pyrotechnic delay material using spectroscopies", Sci Rep, 9, 1 (2019). CrossRef B. S. Lipkin, Picture Processing and Psychopictorics. (Saint Louis, Elsevier Science 2014). DirectLink A. M. Taddese, N. Verrier, M. Debailleul, J.-B. Courbot, and O. Haeberlé, "Optimizing sample illumination scanning in transmission tomographic diffractive microscopy", Appl. Opt., 60, 6 (2021). CrossRef

Partially Coherent Optical Diffraction Tomography Toward Practical Cell Study

Optical diffraction tomography (ODT) is a computational imaging technique based on refractive index (RI) contrast. Its application for microscopic imaging of weakly absorbing and scattering samples has been demonstrated by using a specially designed holographic microscope with angular scanning of the coherent sample illumination direction. Recently, an alternative low cost technique based on partially coherent sample illumination (PC-ODT), which is compatible with the conventional wide-field transmission microscope, has been established. In this case, the 3D refractive index distribution of the sample is obtained by deconvolution from a single stack of through-focus intensity images. The performance of PC-ODT has been successfully tested on various fixed specimens (diatom frustule and biological cells) and moving bacteria. Here, we demonstrate that the PC-ODT is an efficient tool for the analysis of living eukaryotic cell dynamics at short- and long-term periods. The COS-7 cells, which hail from the African green monkey kidney, have been chosen for this study. A fast data acquisition setup comprising an optical scanning module can be easily attached to the microscope, and it allows observing cell 3D organelle movements and RI variations, with the required temporal resolution. In particular, a more rapid nucleoli rotation than previously reported has been found. The long-term cell monitoring during necrosis reveals significant changes in cell dry mass concentration obtained from recovered RI contrast.

Molecular profiling of the intestinal mucosa and immune cells of the colon by multi-parametric histological techniques

The impact of the post-mortem interval (PMI) on the optical molecular characteristics of the colonic mucosa and the gut-associated lymphoid tissue (GALT) were examined by multi-parametric measurements techniques. Inflammatory cells were identified by immunohistochemical staining. Molecular parameters were estimated using the Raman spectroscopy (RS) and Fourier Transform Infrared (FTIR) spectroscopic imaging. The 3D refractive index (3D-RI) distributions of samples were determined using the digital holographic tomography. The distribution of immune cells between post-mortem (PM) and normal controls did show significant differences for CD4 (P = 0.0016) or CD8 (P < 0.0001), whose expression level was decreased in PM cases. No association was found between individual PMI values and inflammatory cell distribution. However, there was a tendency for a negative correlation between CD4+ cells and PMI (r = − 0.542, P = 0.032). The alterations ongoing in post-mortem tissue may suggest that PMI has a suppressive effect on the effector properties of the cell-mediated immunity. Moreover, it was confirmed that spectroscopic and digital holotomographic histology are also a useful technique for characterization of the differences in inflammation of varying intensity and in GALT imaging in a solid tissue. Anatomical location of immune cells and methods of tissue fixation determine the molecular and optical parameters of the examined cases.

Rapid computational cell-rotation around arbitrary axes in 3D with multi-core fiber

Optical trapping is a vital tool in biology, allowing precise optical manipulation of nanoparticles, micro-robots, and cells. Due to the low risk of photodamage and high trap stiffness, fiber-based dual-beam traps are widely used for optical manipulation of large cells. Besides trapping, advanced applications like 3D refractive index tomography need a rotation of cells, which requires precise control of the forces, for example, the acting-point of the forces and the intensities in the region of interest (ROI). A precise rotation of large cells in 3D about arbitrary axes has not been reported yet in dual-beam traps. We introduce a novel dual-beam optical trap in which a multi-core fiber (MCF) is transformed to a phased array, using wavefront shaping and computationally programmable light. The light-field distribution in the trapping region is holographically controlled within 0.1 s, which determines the orientation and the rotation axis of the cell with small retardation. We demonstrate real-time controlled rotation of HL60 cells about all 3D axes with a very high degree of freedom by holographic controlled light through an MCF with a resolution close to the diffraction limit. For the first time, the orientation of the cell can be precisely controlled about all 3D axes in a dual-beam trap. MCFs provide much higher flexibility beyond the bulky optics, enabling lab-on-a-chip applications and can be easily integrated for applications like contactless cell surgery, refractive index tomography, cell-elasticity measurement, which require precise 3D manipulation of cells.

Detection of intracellular monosodium urate crystals in gout synovial fluid using optical diffraction tomography

Optical diffraction tomography (ODT) enables imaging of unlabeled intracellular components by measuring the three-dimensional (3D) refractive index (RI). We aimed to detect intracellular monosodium urate (MSU) crystals in synovial leukocytes derived from gout patients using ODT. The 3D RI values of the synthetic MSU crystals, measured by ODT, ranged between 1.383 and 1.440. After adding synthetic MSU crystals to a macrophage, RI tomograms were reconstructed using ODT, and the reconstructed RI tomograms discerned intracellular and extracellular MSU crystals. We observed unlabeled synthetic MSU crystal entry into the cytoplasm of a macrophage through time-lapse imaging. Furthermore, using gout patient-derived synovial leukocytes, we successfully obtained RI tomogram images of intracellular MSU crystals. The 3D RI identification of MSU crystals was verified with birefringence through polarization-sensitive ODT measurements. Together, our results provide evidence that this novel ODT can identify birefringent MSU crystals in synovial leukocytes of patients with gout.

Bacteria Single-Cell and Photosensitizer Interaction Revealed by Quantitative Phase Imaging.

Quantifying changes in bacteria cells in the presence of antibacterial treatment is one of the main challenges facing contemporary medicine; it is a challenge that is relevant for tackling issues pertaining to bacterial biofilm formation that substantially decreases susceptibility to biocidal agents. Three-dimensional label-free imaging and quantitative analysis of bacteria–photosensitizer interactions, crucial for antimicrobial photodynamic therapy, is still limited due to the use of conventional imaging techniques. We present a new method for investigating the alterations in living cells and quantitatively analyzing the process of bacteria photodynamic inactivation. Digital holographic tomography (DHT) was used for in situ examination of the response of Escherichia coli and Staphylococcus aureus to the accumulation of the photosensitizers immobilized in the copolymer revealed by the changes in the 3D refractive index distributions of single cells. Obtained results were confirmed by confocal microscopy and statistical analysis. We demonstrated that DHT enables real-time characterization of the subcellular structures, the biophysical processes, and the induced local changes of the intracellular density in a label-free manner and at sub-micrometer spatial resolution.

Dynamical machine learning volumetric reconstruction of objects\u2019 interiors from limited angular views

Limited-angle tomography of an interior volume is a challenging, highly ill-posed problem with practical implications in medical and biological imaging, manufacturing, automation, and environmental and food security. Regularizing priors are necessary to reduce artifacts by improving the condition of such problems. Recently, it was shown that one effective way to learn the priors for strongly scattering yet highly structured 3D objects, e.g. layered and Manhattan, is by a static neural network [Goy et al. Proc. Natl. Acad. Sci. 116, 19848–19856 (2019)]. Here, we present a radically different approach where the collection of raw images from multiple angles is viewed analogously to a dynamical system driven by the object-dependent forward scattering operator. The sequence index in the angle of illumination plays the role of discrete time in the dynamical system analogy. Thus, the imaging problem turns into a problem of nonlinear system identification, which also suggests dynamical learning as a better fit to regularize the reconstructions. We devised a Recurrent Neural Network (RNN) architecture with a novel Separable-Convolution Gated Recurrent Unit (SC-GRU) as the fundamental building block. Through a comprehensive comparison of several quantitative metrics, we show that the dynamic method is suitable for a generic interior-volumetric reconstruction under a limited-angle scheme. We show that this approach accurately reconstructs volume interiors under two conditions: weak scattering, when the Radon transform approximation is applicable and the forward operator well defined; and strong scattering, which is nonlinear with respect to the 3D refractive index distribution and includes uncertainty in the forward operator.

Limited-angle tomographic phase microscopy utilizing confocal scanning fluorescence microscopy.

We present a multimodal imaging technique, combining tomographic phase microscopy with limited angular projection range and number, and two-channel spinning-disk confocal scanning fluorescence microscopy. This technique allows high-accuracy 3D refractive index (RI) profiling of live cells in spite of the missing projections. The cellular outer shape and its interior organelles measured by the confocal fluorescence imaging not only specify the cell in molecular levels, but also provide the 3D distributions of the whole cell as well as its organelles. We take these additional 3D morphological details as constraints in Gerchberg-Papoulis-based optical diffraction tomography algorithm. We then obtain an accurate 3D RI tomogram, even with a sparse angular range having a small number of perspective projections, otherwise providing low-accuracy RI reconstruction. Then, we obtain both cellular molecular specificity and inner RI values of the cell and its organelles. We compare the reconstructed 3D RI profiles of various samples, demonstrating the superiority of the proposed technique.

3D reconstruction of weakly scattering objects from 2D intensity-only measurements using the Wolf transform.

A new approach to optical diffraction tomography (ODT) based on intensity measurements is presented. By applying the Wolf transform directly to intensity measurements, we observed unexpected behavior in the 3D reconstruction of the sample. Such a reconstruction does not explicitly represent a quantitative measure of the refractive index of the sample; however, it contains interesting qualitative information. This 3D reconstruction exhibits edge enhancement and contrast enhancement for nanostructures compared with the conventional 3D refractive index reconstruction and thus could be used to localize nanoparticles such as lipids inside a biological sample.

Refractive index distribution of the ice-layer in ICF target from the interference method

In the inertial confinement fusion (ICF) system, the uniformity of the target is important to the successful ignition. A method based on the phase-shifting interferometry and ray tracing is proposed to obtain the 2D (two-dimensional) refractive index distributions of the ice-layer at three wavelengths (532 nm, 785 nm, 1064 nm), from which the 2D density distribution of the ice-layer is got. A differential method is utilized to get the reference refractive index, based on which the refractive index distribution of the layer is retrieved. For targets with uniform refractive index in the ice-layer, simulations show that the relative retrieval accuracies are better than 10 ppb. Experiments show that the refractive index distributions have good consistency at different wavelengths for the same target. The detectable normalized RMS (root mean square) of the refractive index distributions is at least 2.9E-4. The relative accuracies of the average refractive indexes of the shells are better than 1.5% for all three wavelengths. The optimal selection of the two rings on the wavefront map that yields minimal uncertainty to the reference refractive index is discussed. It is concluded that the proposed method and system poses great potential in the in-situ measurement of the refractive index and density distribution of the ICF target layers.

Continuous-wave terahertz diffraction tomography for measuring three-dimensional refractive index maps

Three-dimensional (3D) refractive index (RI) distribution is important to reveal the object’s inner structure. We implemented terahertz (THz) diffraction tomography with a continuous-wave single-frequency THz source for measuring 3D RI maps. The off-axis holographic interference configuration was employed to obtain the quantitative scattered field of the object under each rotation angle. The 3D reconstruction algorithm adopted the filtered backpropagation method, which can theoretically calculate the exact scattering potential from the measured scattered field. Based on the Rytov approximation, the 3D RI distribution of polystyrene foam spheres was achieved with high fidelity, which verified the feasibility of the proposed method.

Quantitative 3D refractive index tomography of opaque samples in epi-mode.

Three-dimensional (3D) refractive index (RI) tomography has recently become an exciting new tool for biological studies. However, its limitation to (1) thin samples resulting from a need of transmissive illumination and (2) small fields of view (typically ~50 μm × 50 μm) has hindered its utility in broader biomedical applications. In this work, we demonstrate 3D RI tomography with a large field of view in opaque, arbitrarily thick scattering samples (unsuitable for imaging with conventional transmissive tomographic techniques) with a penetration depth of ca. one mean free scattering path length (~100 μm in tissue) using a simple, low-cost microscope system with epi-illumination. This approach leverages a solution to the inverse scattering problem via the general non-paraxial 3D optical transfer function of our quantitative oblique back-illumination microscopy (qOBM) optical system. A theoretical analysis is presented along with simulations and experimental validations using polystyrene beads, and rat and human thick brain tissues. This work has significant implications for the investigation of optically thick, semi-infinite samples in a non-invasive and label-free manner. This unique 3D qOBM approach can extend the utility of 3D RI tomography for translational and clinical medicine.

Low-coherence optical diffraction tomography using a ferroelectric liquid crystal spatial light modulator.

Optical diffraction tomography (ODT) is a three-dimensional (3D) label-free imaging technique. The 3D refractive index distribution of a sample can be reconstructed from multiple two-dimensional optical field images via ODT. Herein, we introduce a temporally low-coherence ODT technique using a ferroelectric liquid crystal spatial light modulator (FLC SLM). The fast binary-phase modulation provided by the FLC SLM ensures the high spatiotemporal resolution. To reduce coherent noise, a superluminescent light-emitting diode is used as an economic low-coherence light source. We demonstrate the performance of the proposed system using various samples, including colloidal microspheres and live epithelial cells.

Coarse Raman and optical diffraction tomographic imaging enable label-free phenotyping of isogenic breast cancer cells of varying metastatic potential

Identification of the metastatic potential represents one of the most important tasks for molecular imaging of cancer. While molecular imaging of metastases has witnessed substantial progress as an area of clinical inquiry, determining precisely what differentiates the metastatic phenotype has proven to be more elusive. In this study, we utilize both the morphological and molecular information provided by 3D optical diffraction tomography and Raman spectroscopy, respectively, to propose a label-free route for optical phenotyping of cancer cells at single-cell resolution. By using an isogenic panel of cell lines derived from MDA-MB-231 breast cancer cells that vary in their metastatic potential, we show that 3D refractive index tomograms can capture subtle morphological differences among the parental, circulating tumor cells, and lung metastatic cells. By leveraging its molecular specificity, we demonstrate that coarse Raman microscopy is capable of rapidly mapping a sufficient number of cells for training a random forest classifier that can accurately predict the metastatic potential of cells at a single-cell level. We also perform multivariate curve resolution alternating least squares decomposition of the spectral dataset to demarcate spectra from cytoplasm and nucleus, and test the feasibility of identifying metastatic phenotypes using the spectra only from the cytoplasmic and nuclear regions. Overall, our study provides a rationale for employing coarse Raman mapping to substantially reduce measurement time thereby enabling the acquisition of reasonably large training datasets that hold the key for label-free single-cell analysis and, consequently, for differentiation of indolent from aggressive phenotypes.