Search

Although oxidative stress is believed to play an important role in numerous diseases, current methods of measuring the oxidative stress level do not provide a consensus on how to quantify and compare the level of oxidative stress. Here, we suggest optical diffraction tomography (ODT) as a label-free cellular oxidative stress level quantification method. ODT measurements of HS68 cells treated with hydrogen peroxide solution showed that the oxidative stress level can be quantified based on the biophysical features from the refractive index (RI) tomogram. Quantifying the cellular oxidative stress based on the RI measurements will provide a direct guideline for the level of cell stresses on both academic and clinical situations.

We present a deep learning approach for the rapid resolution enhancement of optical diffraction tomography. Once our three-dimensional U-net-based convolutional neural network learns an image translation between raw tomograms and total-variation-regularized tomograms, the trained network can fill in the missing cone of a measured refractive index tomogram and improve its resolution within seconds. We demonstrate the feasibility and generalizability of our approach on various biological samples, including bacteria, WBC, and NIH3T3.

Three-dimensional (3D), label-free, and long-term imaging in organ-on-a-chip research is necessary to assess the dynamic development of multicellular structures inside a microfluidic chip and relevant physiological functions. However, imaging on-chip multicellular landscapes generally relies on the use of exogenous labels, which limits the long-term imaging of live cells due to photobleaching and phototoxicity. Here, we demonstrate the 3D label-free observation of 5-day on-chip vasculogenesis of human umbilical vein endothelial cells using optical diffraction tomography. The proposed method enables us to quantify both morphological and biochemical alterations during the vasculogenesis.

Optical diffraction tomography (ODT) is a label-free three-dimensional (3D) microscopy technique allowing to retrieve the refractive-index distributions of optically translucent samples. Until recently, the histopathological applications of ODT have been impeded by the difficulty of imaging thick and wide tissues. Here, we demonstrate rapid, wide-field 3D ODT for imaging biological tissues. We designed a high-speed stitching ODT platform exploiting a digital micromirror device and a motorized stage. Developing a fast algorithmic framework that relieves the stitching artifacts and contrast loss due to multiple scattering, we demonstrate high-contrast 3D imaging of 100-m-thick, square-millimeter-wide pathological tissues at sub-micrometer resolution.

Optical diffraction tomography (ODT), which is one of the 3-D imaging techniques, enables non-invasive, label-free, and quantitative imaging of microscopic particles by retrieving multiple optical fields and reconstructing the 3-D hologram image of a sample. However, ODT employing the illumination scanning method suffers from poor axial resolution. In order to remedy this problem, we introduce TOmographic MOuld for optical TRAPping (TOMOTRAP) in ODT, which can control the orientation and shape of arbitrarily shaped dielectric objects. From optical field images of a sample rotated by several angles via TOMOTRAP, we reconstruct the 3-D RI distributions of samples with isotropic spatial resolution.

In coherent imaging systems, parasitic fringes and concentric patterns could commonly be found due to the unwanted multiple reflections [1]. One fundamental solution for the coherent noise is to use a temporally incoherent light source. However, maintaining the full-field interference fringe contrast using temporally incoherent light is not straightforward for interferometric imaging techniques such as quantitative phase imaging (QPI). Fortunately, several brilliant incoherent QPI techniques have been realized for wide-field imaging mainly through the common-path interferometer geometries [2-4]. However, it has been more difficult to implement incoherent-light-based optical diffraction tomography (ODT) due to the additional angle-scanning illumination unit that induces severe decoherence over the camera field-of-view [5]. Here, we suggest a temporally low-coherence optical diffraction tomography by angle-scanning broadband illumination based on general Mach-Zehnder interferometric geometry. We have designed an angle-scanning unit composed of two digital micromirror devices (DMDs) to maintain interference fringe contrast across the whole field of view during the angle-scanning sequence. Further, we have developed the theoretical framework for ODT reconstruction using incoherent light. In the light of our recent developments, we will discuss the theoretical and practical constraints, and suggest the best degree of incoherency for incoherent ODT. We will also demonstrate the incoherent optical diffraction tomography of plastic microspheres, human blood cells and rat pheochromocytoma cells. References 1. I. Choi, K. Lee, and Y. Park, "Compensation of aberration in quantitative phase imaging using lateral shifting and spiral phase integration," Opt. Express 25, 30771-30779 (2017). 2. Z. Wang, L. Millet, M. Mir, H. Ding, S. Unarunotai, J. Rogers, M. U. Gillette, and G. Popescu, "Spatial light interference microscopy (SLIM)," Opt. Express 19, 1016-1026 (2011). 3. B. Bhaduri, H. Pham, M. Mir, and G. Popescu, "Diffraction phase microscopy with white light," Opt. Lett. 37, 1094-1096 (2012). 4. Y. Baek, K. Lee, J. Yoon, K. Kim, and Y. Park, "White-light quantitative phase imaging unit," Opt. Express 24, 9308-9315 (2016). 5. M. Rinehart, Y. Zhu, and A. Wax, "Quantitative phase spectroscopy," Biomed. Opt. Express 3, 958-965 (2012).

First, we present the realization of the ultra-high-definition dynamic 3D holographic display. Using wavefront shaping techniques with highly scattering holo-graphic diffusers, we generate dynamic 3D holographic images with significantly increased viewing angle and image size. Volume speckle fields, generated by holographic diffusers, are actively controlled using a wavefront shaping technique; as a result, the viewing angle and the image size are enhanced by a factor of 2,600 compared to the case of using the DM only. We demonstrate the dynamic display of micrometer-size optical foci in a volume of 8 mm x 8 mm x 20 mm, which far exceeds the capability of existing technology. In addition, we also present a method for direct holography, which enables measurements of an incident field without additional reference beams or assumptions. Inspired from the Siegert equation that connects intensity correlation with field correlations in the time domain, we present the new concept to retrieve the incident field from a speckle intensity pattern in the spatial domain. By measuring a speckle intensity image that is generated when a light field of interest passes through a calibrated turbid layer, field information of an arbitrary optical field can be directly reconstructed. As a proof-of-concept, we demonstrate a compact holographic image sensor, composed of a diffuser and a 2-D image sensor, and present both 2-D and 3-D optical field imaging. [1] Hyeonseung Yu et al., Nature Photonics, 11 :186 (2017) [2] Kyeoreh Lee et al., Nature Communications, 7:13359 (2016)

In this talk, we present correlative approaches in commercial quantitative phase microscopy. By combining both optical diffraction tomography and three-channel 3D fluorescence microscopy, both refractive index tomography and molecular specific imaging of live cells and tissues can be obtained. We will discuss the commercialization of this microscopy (HT-2 series via Tomocube Inc.) and also present potential applications for the study of cell biology and medicine.

In digital 3D holographic displays, the generation of realistic 3D images has been hindered by limited viewing angle and image size. Here we demonstrate a digital 3D holographic display using volume speckle fields produced by scattering layers in which both the viewing angle and the image size are greatly enhanced. Although volume speckle fields exhibit random distributions, the transmitted speckle fields have a linear and deterministic relationship with the input field. By modulating the incident wavefront with a digital micro-mirror device, volume speckle patterns are controlled to generate 3D images of micrometer-size optical foci with 35° viewing angle in a volume of 2 cm × 2 cm × 2 cm.

Imaging brain tissues is an essential part of neuroscience because understanding brain structure provides relevant information about brain functions and alterations associated with diseases. Magnetic resonance imaging and positron emission tomography exemplify conventional brain imaging tools, but these techniques suffer from low spatial resolution around 100 μm. As a complementary method, histopathology has been utilized with the development of optical microscopy. The traditional method provides the structural information about biological tissues to cellular scales, but relies on labor-intensive staining procedures. With the advances of illumination sources, label-free imaging techniques based on nonlinear interactions, such as multiphoton excitations and Raman scattering, have been applied to molecule-specific histopathology. Nevertheless, these techniques provide limited qualitative information and require a pulsed laser, which is difficult to use for pathologists with no laser training. Here, we present a label-free optical imaging of mouse brain tissues for addressing structural alteration in Alzheimer’s disease. To achieve the mesoscopic, unlabeled tissue images with high contrast and sub-micrometer lateral resolution, we employed holographic microscopy and an automated scanning platform. From the acquired hologram of the brain tissues, we could retrieve scattering coefficients and anisotropies according to the modified scattering-phase theorem. This label-free imaging technique enabled direct access to structural information throughout the tissues with a sub-micrometer lateral resolution and presented a unique means to investigate the structural changes in the optical properties of biological tissues.

Holographic optical tweezers (HOTs) have been utilized for trapping microscopic particles in three dimensions with multiple foci generated by wavefront shaping of light, which can manipulate three-dimensional (3-D) positions of colloidal particles as well as exerting an optical force on particles. So far, most experiments using HOTs have been conducted for trapping spherical particles because optical principles can easily predict optical forces and the responding motion of microspheres. For non-spherical particles, however, calculation of optical forces and torques exerting on samples is very complicated, and the orientation control of non-spherical particles is limited since the non-spherical particles tend to align along the optic axis of the trapping beam. Here, we propose and experimentally demonstrate 3-D trapping of non-spherical particles by wavefront shaping of light based on the measurement of 3-D refractive index (RI) distribution of samples. The 3-D RI distribution of non-spherical particles was measured by optical diffraction tomography and the phase hologram which can generate stable optical traps for the samples was calculated by iterative 3-D Gerchberg-Saxton algorithm from the measured 3-D RI distribution. We first validate the proposed method for stable trapping and orientation control of 2-m colloidal PMMA ellipsoids. The proposed method is also exploited for rotating, folding and assembly of red blood cells.

There is a strong correlation between the dynamic membrane fluctuations and the biomechanical properties of living cells. The dynamic membrane fluctuation consists of submicron displacements, and can be altered by changing the cells’ pathophysiological conditions. These results have significant relevance to the understanding of RBC biophysics and pathology, as follows. RBCs must withstand large mechanical deformations during repeated passages through the microvasculature and the fenestrated walls of the splenic sinusoids. This essential ability is diminished with senescence, resulting in physiological destruction of the aging RBCs. Pathological destruction of the red cells, however, occurs in cells affected by a host of diseases such as spherocytosis, malaria, and Sickle cell disease, as RBCs depart from their normal discoid shape and lose their deformability. Therefore, quantifying the RBC deformability insight into a variety of problems regarding the interplay of cell structure, dynamics, and function. Furthermore, the ability to monitor mechanical properties of RBCs is of vital interest in monitoring disease progression or response to treatment as molecular and pharmaceutical approaches for treatment of chronic diseases. Here, we present the measurements of dynamic membrane fluctuations in live cells using quantitative phase imaging techniques. Measuring both the 3-D refractive index maps and the dynamic phase images of live cells are simultaneously measured, from which dynamic membrane fluctuation and deformability of cells are precisely calculated. We also present its applications to various diseases ranging from sickle cell diseases, babesiosis, and to diabetes.

Optical diffraction tomography (ODT) has been an emerging optical technique for label-free imaging of three-dimensional (3-D) refractive index (RI) distribution of biological samples. ODT employs interferometric microscopy for measuring multiple holograms of samples with various incident angles, from which the Fourier diffraction theorem reconstructs the 3-D RI distribution of samples from retrieved complex optical fields. Since the RI value is linearly proportional to the protein concentration of biological samples where the proportional coefficient is called as refractive index increment (RII), reconstructed 3-D RI tomograms provide precise structural and biochemical information of individual biological samples. Because most proteins have similar RII value, however, ODT has limited molecular specificity, especially for imaging eukaryotic cells having various types of proteins and subcellular organelles. Here, we present an ODT system combined with structured illumination microscopy which can measure the 3-D RI distribution of biological samples as well as 3-D super-resolution fluorescent images in the same optical setup. A digital micromirror device (DMD) controls the incident angle of the illumination beam for tomogram reconstruction, and the same DMD modulates the structured illumination pattern of the excitation beam for super-resolution fluorescent imaging. We first validate the proposed method for simultaneous optical diffraction tomographic imaging and super-resolution fluorescent imaging of fluorescent beads. The proposed method is also exploited for various biological samples.

Using a common-path interferometric technique, we measure biomechanical and morphological properties of individual red blood cells in SCD patients as a function of cell density, and investigate the correlation of these biophysical properties with drug intake as well as other clinically measured parameters.

When a light propagates through highly disordered medium, its optical parameters such as amplitude, phase and polarization states are completely scrambled because of multiple scattering events. Since the multiple scattering is a fundamental optical process that contains extremely high degrees of freedom, optical information of a transmitted light is totally mingled. Until recently, the presence of multiple scattering in an inhomogeneous medium is considered as a major obstacle when manipulating a light transmitting through the medium. However, a recent development of wavefront shaping techniques enable us to control the propagation of light through turbid media; a light transmitting through a turbid medium can be effectively controlled by modulating the spatial profile of the incident light using spatial light modulator. In this work, stand-alone scattering optical device is proposed; a holographic photopolymer film, which is much economic compared to the other digital spatial light modulators, is used to record and reconstruct permanent wavefront to generate optical field behind a scattering medium. By employing our method, arbitrary optical field can be generated since the scattering medium completely mixes all the optical parameters which allow us to access all the optical information only by modulating spatial phase profile of the impinging wavefront. The method is experimentally demonstrated in both the far-field and near-field regime where it shows promising fidelity and stability. The proposed stand-alone scattering optical device will opens up new avenues for exploiting the randomness inherent in disordered medium.

White blood cells (WBC) have crucial roles in immune systems which defend the host against from disease conditions and harmful invaders. Various WBC subsets have been characterized and reported to be involved in many pathophysiologic conditions. It is crucial to isolate a specific WBC subset to study its pathophysiological roles in diseases. Identification methods for a specific WBC population are rely on invasive approaches, including Wright-Gimesa staining for observing cellular morphologies and fluorescence staining for specific protein markers. While these methods enable precise classification of WBC populations, they could disturb cellular viability or functions. In order to classify WBC populations in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a three-dimensional (3-D) quantitative phase imaging technique that measures 3-D refractive index (RI) distributions of individual WBCs. To test feasibility of label-free classification of WBC populations using ODT, we measured four subtypes of WBCs, including B cell, CD4 T cell, CD8 T cell, and natural killer (NK) cell. From measured 3-D RI tomograms of WBCs, we obtain quantitative structural and biochemical information and classify each WBC population using a machine learning algorithm.

Parkinson’s disease (PD) is a common neurodegenerative disease that causes symptoms of postural instability and slowness of movement. Neurodegeneration in dopaminergic neurons at the substantia nigra has been reported as pathologic features, however, detailed mechanisms underlying neurodegeneration are still remain unclear. To investigate a neurodegenerative process, various imaging tools including phase contrast microscopy, electron microscopy, and fluorescence microscopy are utilized. However, these imaging methods provide qualitative information and require invasive approaches such as the use of fluorescence agents or chemical fixation procedures that disturb normal physiological conditions of neuron cells. In order to quantify the neurodegenerative process in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a 3D quantitative phase imaging method that measures 3D refractive index (RI) distributions of a sample which provide quantitative structural (volume, surface area, sphericity) and biochemical (protein concentration, total cellular dry mass) information. We investigated neurotoxic effects of MPP+ on SH-SY5Y cells by using quantitative information obtained from 3D RI distributions. We also performed temporal measurements of 3D RI distributions of an individual SH-SY5Y cell to analyze neurotoxic effects on intracellular vesicle dynamics.

We systematically measure the morphological, biochemical, and biomechanical properties of individual human red blood cells (RBCs) from patients with diabetes mellitus using quantitative phase imaging technique to characterize the diabetic red cells with respect to those of the healthy. The 3-D refractive index tomograms and 2-D dynamic membrane fluctuation maps of individual RBCs are reconstructed from a set of the retrieved complex optical fields at various laser incidence angles using the Common-path diffraction optical tomography, from which volume, surface area, sphericity, hemoglobin (Hb) concentration, Hb content, and membrane fluctuation are obtained simultaneously. The correlative relations among the retrieved red cell indices of diabetic and healthy RBCs are also investigated with capabilities of individual cell measurement. As expected, there are no significant alterations in morphologies (cellular volumes, surface area, and sphericity) between diabetic and healthy RBCs. However, despite the minute mean corpuscular Hb differences in cell blood count datasheet, the measured Hb concentrations and Hb contents of diabetic RBCs are statistically higher than those of healthy RBCs, which might be related to the glycation of Hb molecules by hyperglycemia. Meanwhile, the membrane fluctuations of diabetic RBCs are clearly diminished compared to healthy red cells, implying the significantly decreased RBC deformability. In particular, it seems that the membrane fluctuations have mild negative relationships with the reported HbA1c levels.

It is commonly known that alteration of erythrocyte deformability lead to serious microcirculatory diseases such as retinopathy, nephropathy, etc. Various methods and technologies have been developed to diagnose such membrane properties of erythrocytes. In this study, we developed an innovative method to measure hemorheological characteristics of the erythrocyte membrane using a light scattering analysis with simplified optic setting and multi-cell analysis as well. Light scattering intensity through multiple erythrocytes and its power density spectrum were obtained. The results of light scattering analyses were compared in healthy control and artificially hardened sample which was treated with glutaraldehyde. These results were further compared with conventional assays to measure deformable property in hemorheology. We found that light scattering information would reflect the disturbance of membrane fluctuation in artificially damaged erythrocytes. Therefore, measuring fluctuation of erythrocyte membrane using light scattering signal could facilitate simple and precise diagnose of pathological state on erythrocyte as well as related complications.

Multiple scattering is a significant obstacle in the optical imaging of biological samples. However, it is possible to reverse its effects through optical phase conjugation (OPC) of the scattered field. We perform digital OPC (DOPC) utilizing a spatial light modulator (SLM) and a Sagnac interferometer geometry. This design permits a simple and robust DOPC implementation, which we demonstrate experimentally. We exploit the beam-shaping flexibility of the SLM to demonstrate the possibility to enhance either the optical power transmission or the light focusing ability of the DOPC process.

The use of coherent light in conventional holographic phase microscopy (HPM) poses three major drawbacks: poor spatial resolution, weak depth sectioning, and fixed pattern noise due to unwanted diffraction. Here, we report a technique which can overcome these drawbacks, but maintains the advantage of phase microscopy - high contrast live cell imaging and 3D imaging. A speckle beam of a complex spatial pattern is used for illumination to reduce fixed pattern noise and to improve optical sectioning capability. By recording of the electric field of speckle, we demonstrate high contrast 3D live cell imaging without the need for axial scanning - neither objective lens nor sample stage. This technique has great potential in studying biological samples with improved sensitivity, resolution and optical sectioning capability.

We report a practical method for label-free quantification of specific molecules using spectroscopic imaging of sample-induced phase shifts (for the detail, please see the Ref. [1]). Diffraction phase microscopy equipped with various wavelengths of light source is used to record wavelength-dependent phase images. We first perform dispersion measurements on pure solutions of single molecular species present in the cells, such as albumin and hemoglobin (Hb). With this prior calibration of molecular specific dispersion, we demonstrate the extraction of Hb concentration from individual red blood cells (RBCs). The end point of this study is non-invasive monitoring of physiological states of intact living cells.