The remarkable deformability of the human red blood cell (RBC) results from the coupled dynamic response of the phospholipid bilayer and the spectrin molecular network. Here we present quantitative connections between spectrin morphology and membrane fluctuations of human RBCs by using dynamic full-field laser interferometry techniques. We present conclusive evidence that the presence of adenosine 5′-triphosphate (ATP) facilitates nonequilibrium dynamic fluctuations in the RBC membrane that are highly correlated with the biconcave shape of RBCs. Spatial analysis of the fluctuations reveals that these non-equilibrium membrane vibrations are enhanced at the scale of spectrin mesh size. Our results indicate that the dynamic remodeling of the coupled membranes powered by ATP results in non-equilibrium membrane fluctuations manifesting from both metabolic and thermal energies and also maintains the biconcave shape of RBCs.ATP | imaging technique | membrane fluctuation | RBC | spectrin A s they travel through small blood vessels and organs, RBCs undergo repeated severe deformation. The coupling and interactions between the phospholipid bilayer and the spectrin network govern the deformability of RBCs (1). The fluid-like lipid bilayer is coupled to the two-dimensional spectrin network that comprises an approximately hexagonal lattice via protein junctional complexes. The RBC membrane is remarkably soft and elastic, and thus exhibits fluctuations with amplitudes of the order of tens of nanometers. The dynamics of the RBC membrane is strongly related to the membrane structure and mechanical properties and has been explored extensively (2-6). However, experimental results available to date on RBC membrane fluctuations have provided only limited information on select regions of the cell membrane with limited spatial and/or temporal resolution (7-9). No full-field measurements of membrane fluctuations in the entire RBC arising in response to well-controlled metabolic activity have been made so far and, consequently, different techniques have led to different interpretations of the mechanistic origins of dynamic RBC membrane fluctuations with and without metabolic activity (7-9).The RBC membrane is not a static but a metabolically regulated active structure. It is known that biochemical energy controls its static and dynamic characteristics. The presence of ATP is not only crucial in maintaining the biconcave shape of the RBC membrane (10), but was also shown to increase the dynamic membrane fluctuations (7, 9). However, the regulatory mechanism of ATP in RBC membranes still remains elusive. Furthermore, these static and dynamic effects of ATP on RBC membrane fluctuations have hitherto been regarded as separate phenomena and have never been explored simultaneously.Here, we present dynamic, full-field, and quantitative measurements of ATP effects on RBC membrane morphology and fluctuations. We show that in the presence of ATP, the RBC membrane fluctuations have a non-equilibrium, metabolic component in addition to a thermal o...

A cellular-level study of the pathophysiology is crucial for understanding the mechanisms behind human diseases. Recent advances in quantitative phase imaging (QPI) techniques show promises for the cellular-level understanding of the pathophysiology of diseases. To provide important insight on how the QPI techniques potentially improve the study of cell pathophysiology, here we present the principles of QPI and highlight some of the recent applications of QPI ranging from cell homeostasis to infectious diseases and cancer.

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.

Biologically driven nonequilibrium fluctuations are often characterized by their non-Gaussianity or by an "effective temperature", which is frequency dependent and higher than the ambient temperature. We address these two measures theoretically by examining a randomly kicked particle, with a variable number of kicking motors, and show how these two indicators of nonequilibrium behavior can contradict. Our results are compared with new experiments on shape fluctuations of red-blood cell membranes, and demonstrate how the physical nature of the motors in this system can be revealed using these global measures of nonequilibrium.

We have developed diffraction phase and fluorescence (DPF) microscopy as a new technique for simultaneous quantitative phase imaging and epi-fluorescence investigation of live cells. The DPF instrument consists of an interference microscope, which is incorporated into a conventional inverted fluorescence microscope. The quantitative phase images are characterized by sub-nanometer optical path-length stability over periods from milliseconds to a cell lifetime. The potential of the technique for quantifying rapid nanoscale motions in live cells is demonstrated by experiments on red blood cells, while the composite phase-fluorescence imaging mode is exemplified with mitotic kidney cells.

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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Due to its strong correlation with the pathophysiology of many diseases, information about human red blood cells (RBCs) has a crucial function in hematology. Therefore, measuring and understanding the morphological, chemical, and mechanical properties of individual RBCs is a key to understanding the pathophysiology of a number of diseases in hematology, as well as to opening up new possibilities for diagnosing diseases in their early stages. In this study, we present the simultaneous and quantitative measurement of the morphological, chemical, and mechanical parameters of individual RBCs employing optical holographic microtomography. In addition, it is demonstrated that the correlation analyses of these RBC parameters provide unique information for distinguishing and understanding diseases.