Caenorhabditis elegans can survive upon harsh environments by entering dauer diapause with reduced metabolic activity and distinctive structural changes. We employed optical diffraction tomography (ODT) to quantitatively measure the transition of mass density distribution of living C. elegans larvae in the reproductive and diapause stages. ODT revealed that the mass density of C. elegans larvae increased upon entry into dauer diapause, and surprisingly, the harshly desiccated dauer larvae exhibited very high refractive index values (n ~ 1.5). Moreover, mutants that are sensitive to desiccation displayed structural abnormalities in the anhydrobiotic stage that were not observed by conventional microscopy.
Achieving the isotropic 3D resolution has been one of the most challenging in optical microscopy. When objective lenses with finite numerical aperture, their axial resolution becomes inferior to the lateral resolution. Although various sample rotation methods have been demonstrated to improve the axial resolution, the requirement of invasive sample manipulations has limited their applications for general complex-shaped specimens. Here, we propose a general method for the in-situ isotropic microtomography of freestanding specimens. Exploiting complex wavefront shaping and optical tweezers, we demonstrate that optimally structured 3D light traps can stably rotate a specimen by considering their 3D refractive index distribution, and reconstruct tomograms with isotropic resolution.
We present a combined optical system for Brillouin microscopy with optical diffraction tomography (ODT), which can reconstruct the three-dimensional refractive index (RI) distribution of biological samples. By correlating Brillouin frequency shift with the reconstructed RI distribution in the same field-of-view, we can calculate the precise longitudinal modulus of the samples without a priori information. We demonstrate the capabilities of the method using a cell phantom consisting of different hydrogel beads with known mechanical properties, and apply it to the quantitative characterization of the mechanical and biophysical properties of nuclear compartments inside individual cells under various physiological conditions including cell cycle progression and drug treatments.
The viscoelastic material properties of biological systems are increasingly recognized as important parts of signaling cascades involved in developmental and pathological processes. They are furthermore assumed to play a crucial role in surviving extreme environmental conditions for certain organisms, such as yeast cells. Confocal Brillouin microscopy gives access to the viscoelastic material properties of single cells and tissues in a contact- and label-free manner and with a high spatial resolution. In combination with quantitative phase imaging, it is then possible to determine the longitudinal modulus and the viscosity of the sample. In this study, we probed living zebrafish larvae in all anatomical planes, at different time points during development and after spinal cord injury. We could show, that confocal Brillouin microscopy detects the viscoelasticity of different anatomical structures without affecting the animal’s development. We furthermore observed a transiently decreasing Brillouin shift after spinal cord injury and a difference in Brillouin shift between in vivo and ex vivo measurements of the same sample region. Using quantitative phase imaging we additionally show, that the Brillouin shift of the probed tissues is mainly governed by their longitudinal modulus and viscosity. In conclusion, this work constitutes the methodical basis to identify key determinants of viscoelastic tissue properties during biologically important processes in vivo.
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.
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.
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.
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.
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.
Optical diffraction tomography (ODT) is an interferometric microscopy technique capable of measuring 3-D refractive index (RI) distribution of transparent samples. Multiple 2-D holograms of a sample illuminated with various angles are measured, from which 3-D RI map of the sample is reconstructed via the diffraction theory. ODT has been proved as a powerful tool for the study of biological cells, due to its non-invasiveness, label-free and quantitative imaging capability. Recently, our group has demonstrated that a digital micromirror device (DMD) can be exploited for fast and precise control of illumination beams for ODT. In this work, we systematically study the precision and stability of the ODT system equipped with a DMD and present measurements of 3-D and 4-D RI maps of various types of live cells including human red blood cells, white blood cells, hepatocytes, and HeLa cells. Furthermore, we also demonstrate the effective visualization of 3-D RI maps of live cells utilizing the measured information about the values and gradient of RI tomograms.
Red blood cells (RBCs) from the cord blood of newborn infants have distinctive functions in fetal and infant development. To systematically investigate the biophysical characteristics of individual cord RBCs in newborn infants, a comparative study was performed on RBCs from the cord blood of newborn infants and from adult mothers or nonpregnant women using optical holographic microtomography. Optical measurements of the distributions of the three-dimensional refractive indices and the dynamic membrane fluctuations of individual RBCs were used to investigate the morphological, biochemical, and mechanical properties of cord, maternal, and adult RBCs at the individual cell level. The volume and surface area of the cord RBCs were significantly larger than those of the RBCs from nonpregnant women, and the cord RBCs had more flattened shapes than that of the RBCs in adults. In addition, the hemoglobin (Hb) content in the cord RBCs from newborns was significantly higher. The Hb concentration in the cord RBCs was higher than that in the nonpregnant women or maternal RBCs, but they were within the physiological range of adults. Interestingly, the amplitudes of the dynamic membrane fluctuations in cord RBCs were comparable to those in nonpregnant women and maternal RBCs, suggesting that the deformability of cord RBCs is similar to that of healthy RBCs in adults.
We present optical measurements of morphology and refractive indexes (RIs) of human downy arm hairs using three-dimensional (3-D) quantitative phase imaging techniques. 3-D RI tomograms and high-resolution two-dimensional synthetic aperture images of individual downy arm hairs were measured using a Mach–Zehnder laser interferometric microscopy equipped with a two-axis galvanometer mirror. From the measured quantitative images, the RIs and morphological parameters of downy hairs were noninvasively quantified including the mean RI, volume, cylinder, and effective radius of individual hairs. In addition, the effects of hydrogen peroxide on individual downy hairs were investigated.
Common-path diffraction optical tomography (cDOT) is a non-invasive and label-free optical holographic technique for measuring both the three-dimensional refractive index (RI) tomograms and two-dimensional dynamic phase images of a sample. Due to common-path geometry, cDOT provides quantitative phase imaging with high phase sensitivity. However, the image quality of the cDOT suffers from speckle noise; the use of a monochromatic laser inevitably results in the formation of parasitic fringe patterns in measured quantitative phase images. Here, we present a technique to reduce speckle noise in the cDOT using a low-coherence illumination source. Utilizing a Ti-sapphire pulsed laser in the cDOT, we achieved the reduction of speckle noise in both the three-dimensional RI tomograms and two-dimensional dynamic phase images.
We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated.
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