Abstract. Three-dimensional imaging of biological cells is crucial for the investigation of cell biology, providing valuable information to reveal the mechanisms behind pathophysiology of cells and tissues. Recent advances in optical diffraction tomography (ODT) have demonstrated the potential for the study of various cells with its unique advantages of quantitative and label-free imaging capability. To provide insight on this rapidly growing field of research and to discuss its applications in biology and medicine, we present the summary of the ODT principle and highlight recent studies utilizing ODT with the emphasis on the applications to the pathophysiology of cells. Stephens, and V. J. Allan, "Light microscopy techniques for live cell Imaging," Science 300, 82-86 (2003). 2. M. Minsky, "Microscopy apparatus," US 3, 013,467 (Dec. 19 1961). 3. W. Denk, J. P. Strickler, and W. W. Webb, "Two-photon laser microscopy," US 5,034,613 (Jul. 23 1991). 4. B. Huang, M. Bates, and X. Zhuang, "Super resolution fluorescence microscopy," Annual Review of Biochemistry 78, 993-1016Biochemistry 78, 993- (2009. 5. E. Wolf, "Three-dimensional structure determination of semi-transparent objects from holographic data,"Optics Communications 1, 153-156 (1969). 6. R. Dändliker, and K. Weiss, "Reconstruction of the three-dimensional refractive index from scattered waves,"Optics Communications 1(7), 323-328 (1970 2427-2439 (1979). 8. N. Streibl, "Three-dimensional imaging by a microscope," Journal of the Optical Society of America A 2(2), 121-127 (1985). 9. S. Kawata, O. Nakamura, and S. Minami, "Optical Microscope Tomography .1. Support Constraint," Journal of the Optical Society of America A 4(1), 292-297 (1987). 10. T. Noda, S. Kawata, and S. Minami, "Three-dimensional phase contrast imaging by an annular illumination microscope," Applied Optics 29(26), 3810-3815 (1990). 11. A. J. Devaney, and A. Schatzberg, "Coherent optical tomographic microscope," Proc. of SPIE 1767SPIE , 62-71 (1992. 12. G. Vishnyakov and G. Levin, "Optical microtomography of phase objects," Optics and Spectroscopy 85(1), 73-77 (1998 tomography with a low-coherence illumination for reducing speckle noise," Proc. of SPIE 9336, 933629 (2015).

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We present a powerful and cost-effective method for active illumination using a digital micromirror device (DMD) for quantitative phase imaging techniques. Displaying binary illumination patterns on a DMD with appropriate spatial filtering, plane waves with various illumination angles are generated and impinged onto a sample. Complex optical fields of the sample obtained with various incident angles are then measured via Mach-Zehnder interferometry, from which a high-resolution two-dimensional synthetic aperture phase image and a three-dimensional refractive index tomogram of the sample are reconstructed. We demonstrate the fast and stable illumination control capability of the proposed method by imaging colloidal spheres and biological cells, including a human red blood cell and a HeLa cell.Quantitative phase imaging (QPI) has emerged as an invaluable tool for imaging small transparent objects, such as biological cells and tissues [1,2]. QPI employs various interferometric microscopy techniques, including quantitative phase microscopy [2] and digital holographic microscopy [3], to quantitatively measure the optical phase delay of samples. In particular, the measured optical phase delay provides information about the morphological and biochemical properties of biological samples at the single-cell level. Recently, QPI techniques have been widely applied to study the pathophysiology of various biological cells and tissues, including red blood cells (RBCs) [4][5][6][7], white blood cells [8], bacteria [9][10][11], neurons [12][13][14], and cancer cells [15,16].Controlling the illumination beam is crucial in QPI. Especially for measuring 3-D refractive index (RI) tomograms [17] or 2-D highresolution synthetic aperture images [18], angles of plane wave illumination impinging onto a sample should be systematically controlled, and the corresponding light field images of the sample should be measured. Traditionally, galvanometer-based rotating mirrors have been used to control the angle of the illumination beam. A galvanometer-based rotating mirror located at the plane that is conjugate to a sample can control the angle of the incident beam by tilting the mirror with a certain electric voltage.The use of galvanometers, however, has several disadvantages. Inherently, there exists mechanical instability due to position jittering induced by electric noise and positioning error at high voltages. When a two-axis galvanometer is used, the rotational surfaces of each axis cannot be simultaneously conjugate to a sample due to its geometry, and this may induce unwanted additional quadratic phase distribution on the illumination beam that can limit the accurate measurements of 3-D RI tomograms. To solve this optical misalignment, two one-axis galvanometers can be placed at separate conjugate planes, but it requires a bulky optical setup with a long optical path, which can deteriorate phase noise. More importantly, galvanometers cannot generate acomplex wavefront; only tilting of a plane wave is permitted. Recently, a spatial lig...

Lipid droplets (LDs) are subcellular organelles with important roles in lipid storage and metabolism and involved in various diseases including cancer, obesity, and diabetes. Conventional methods, however, have limited ability to provide quantitative information on individual LDs and have limited capability for three-dimensional (3-D) imaging of LDs in live cells especially for fast acquisition of 3-D dynamics. Here, we present an optical method based on 3-D quantitative phase imaging to measure the 3-D structural distribution and biochemical parameters (concentration and dry mass) of individual LDs in live cells without using exogenous labelling agents. The biochemical change of LDs under oleic acid treatment was quantitatively investigated, and 4-D tracking of the fast dynamics of LDs revealed the intracellular transport of LDs in live cells.

Two-dimensional (2D) nanomaterials, such as graphene-based materials and transition metal dichalcogenide (TMD) nanosheets, are promising materials for biomedical applications owing to their remarkable cytocompatibility and physicochemical properties. On the basis of their potent antibacterial properties, 2D materials have potential as antibacterial films, wherein the 2D nanosheets are immobilized on the surface and the bacteria may contact with the basal planes of 2D nanosheets dominantly rather than contact with the sharp edges of nanosheets. To address these points, in this study, we prepared an effective antibacterial surface consisting of representative 2D materials, i.e., graphene oxide (GO) and molybdenum disulfide (MoS), formed into nanosheets on a transparent substrate for real device applications. The antimicrobial properties of the GO-MoS nanocomposite surface toward the Gram-negative bacteria Escherichia coli were investigated, and the GO-MoS nanocomposite exhibited enhanced antimicrobial effects with increased glutathione oxidation capacity and partial conductivity. Furthermore, direct imaging of continuous morphological destruction in the individual bacterial cells having contacts with the GO-MoS nanocomposite surface was characterized by holotomographic (HT) microscopy, which could be used to detect the refractive index (RI) distribution of each voxel in bacterial cell and reconstruct the three-dimensional (3D) mapping images of bacteria. In this regard, the decreases in both the volume (67.2%) and the dry mass (78.8%) of bacterial cells that came in contact with the surface for 80 min were quantitatively measured, and releasing of intracellular components mediated by membrane and oxidative stress was observed. Our findings provided new insights into the antibacterial properties of 2D nanocomposite film with label-free tracing of bacterial cell which improve our understanding of antimicrobial activities and opened a window for the 2D nanocomposite as a practical antibacterial film in biomedical applications.

Identification of lymphocyte cell types are crucial for understanding their pathophysiological roles in human diseases. Current methods for discriminating lymphocyte cell types primarily rely on labelling techniques with magnetic beads or fluorescence agents, which take time and have costs for sample preparation and may also have a potential risk of altering cellular functions. Here, we present the identification of non-activated lymphocyte cell types at the single-cell level using refractive index (RI) tomography and machine learning. From the measurements of three-dimensional RI maps of individual lymphocytes, the morphological and biochemical properties of the cells are quantitatively retrieved. To construct cell type classification models, various statistical classification algorithms are compared, and the k-NN (k = 4) algorithm was selected. The algorithm combines multiple quantitative characteristics of the lymphocyte to construct the cell type classifiers. After optimizing the feature sets via cross-validation, the trained classifiers enable identification of three lymphocyte cell types (B, CD4+ T, and CD8+ T cells) with high sensitivity and specificity. The present method, which combines RI tomography and machine learning for the first time to our knowledge, could be a versatile tool for investigating the pathophysiological roles of lymphocytes in various diseases including cancers, autoimmune diseases, and virus infections.

Abstract:The characterization of white blood cells (WBCs) is crucial for blood analyses and disease diagnoses. However, current standard techniques rely on cell labeling, a process which imposes significant limitations. Here we present three-dimensional (3D) optical measurements and the label-free characterization of mouse WBCs using optical diffraction tomography. 3D refractive index (RI) tomograms of individual WBCs are constructed from multiple two-dimensional quantitative phase images of samples illuminated at various angles of incidence. Measurements of the 3D RI tomogram of WBCs enable the separation of heterogeneous populations of WBCs using quantitative morphological and biochemical information. Time-lapse tomographic measurements also provide the 3D trajectory of micrometer-sized beads ingested by WBCs. These results demonstrate that optical diffraction tomography can be a useful and versatile tool for the study of WBCs. References and links1. L. Balagopalan, E. Sherman, V. A. Barr, and L. E. Samelson, "Imaging techniques for assaying lymphocyte activation in action," Nat. Rev. Immunol. 11(1), 21-33 (2011). 2. J. C. Edwards and G. Cambridge, "B-cell targeting in rheumatoid arthritis and other autoimmune diseases," Nat.Rev. Immunol. 6(5), 394-403 (2006). 3. M. T. Heneka, M. P. Kummer, and E. Latz, "Innate immune activation in neurodegenerative disease," Nat. Rev.Immunol. 14(7), 463-477 (2014). 4. W. Zou and N. P. Restifo, "T(H)17 cells in tumour immunity and immunotherapy," Nat. Rev. Immunol. 10(4), 248-256 (2010). 41. K. E. Handwerger, J. A. Cordero, and J. G. Gall, "Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure," Mol.

We present an optical holographic micro-tomographic technique for imaging both the three-dimensional structures and dynamics of biological cells. Optical light field images of a sample, illuminated by a plane wave with various illumination angles, are measured in a common-path interferometry, and thus both the three-dimensional refractive index tomogram and two-dimensional dynamics of live biological cells are measured with extremely high sensitivity. The applicability of the technique is demonstrated through quantitative and measurements of morphological, chemical, and mechanical parameters at the individual cell level.