Source: https://www.osapublishing.org/oe/abstract.cfm?uri=oe-17-22-19674
Timestamp: 2019-04-24 15:51:59+00:00

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This report presents a model-independent method of assessing contributions to the light scattering from individual organelles in single intact cells. We first measure the 3D index map of a living cell, and then modify the map in such a way so as to eliminate contrast due to a particular intracellular organelle. By calculating and comparing the light scattering distributions calculated from the original and modified index maps using the Rytov approximation, we extract the light scattering contribution from the particular organelle of interest. The relative contributions of the nucleus and nucleolus to the scattering of the entire cell are thus determined, and the applicability of the homogeneous spherical model to non-spherical and heterogeneous organelles in forward scattering is evaluated.
V. Backman, R. Gurjar, K. Badizadegan, L. Itzkan, R. R. Dasari, L. T. Perelman, and M. S. Feld, “Polarized light scattering spectroscopy for quantitative measurement of epithelial cellular structures in situ,” IEEE J. Sel. Top. Quantum Electron. 5(4), 1019–1026 (1999).
C. Mujat, C. Greiner, A. Baldwin, J. M. Levitt, F. Tian, L. A. Stucenski, M. Hunter, Y. L. Kim, V. Backman, M. Feld, K. Münger, and I. Georgakoudi, “Endogenous optical biomarkers of normal and human papillomavirus immortalized epithelial cells,” Int. J. Cancer 122(2), 363–371 (2008).
H. Fang, M. Ollero, E. Vitkin, L. M. Kimerer, P. B. Cipolloni, M. M. Zaman, S. D. Freedman, I. J. Bigio, I. Itzkan, E. B. Hanlon, and L. T. Perelman, “Noninvasive sizing of subcellular organelles with light scattering spectroscopy,” IEEE J. Sel. Top. Quantum Electron. 9(2), 267–276 (2003).
R. S. Gurjar, V. Backman, L. T. Perelman, I. Georgakoudi, K. Badizadegan, I. Itzkan, R. R. Dasari, and M. S. Feld, “Imaging human epithelial properties with polarized light-scattering spectroscopy,” Nat. Med. 7(11), 1245–1248 (2001).
M. Hunter, V. Backman, G. Popescu, M. Kalashnikov, C. W. Boone, A. Wax, V. Gopal, K. Badizadegan, G. D. Stoner, and M. S. Feld, “Tissue self-affinity and polarized light scattering in the born approximation: A new model for precancer detection,” Phys. Rev. Lett. 97(13), 138102 (2006).
J. R. Mourant, T. M. Johnson, S. Carpenter, A. Guerra, T. Aida, and J. P. Freyer, “Polarized angular dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures,” J. Biomed. Opt. 7(3), 378–387 (2002).
A. Wax, C. Yang, V. Backman, K. Badizadegan, C. W. Boone, R. R. Dasari, and M. S. Feld, “Cellular Organization and Substructure Measured Using Angle-Resolved Low-Coherence Interferometry,” Biophys. J. 82(4), 2256–2264 (2002).
A. Wax, C. Yang, M. G. Müller, R. Nines, C. W. Boone, V. E. Steele, G. D. Stoner, R. R. Dasari, and M. S. Feld, “In Situ Detection of Neoplastic Transformation and Chemopreventive Effects in Rat Esophagus Epithelium Using Angle-resolved Low-coherence Interferometry,” Cancer Res. 63(13), 3556–3559 (2003).
J. D. Wilson and T. H. Foster, “Mie theory interpretations of light scattering from intact cells,” Opt. Lett. 30(18), 2442–2444 (2005).
M. Xu, T. T. Wu, and J. A. Y. Qu, “Unified Mie and fractal scattering by cells and experimental study on application in optical characterization of cellular and subcellular structures,” J. Biomed. Opt. 13, (2008).
C. C. Yu, C. Lau, J. W. Tunnell, M. Hunter, M. Kalashnikov, C. Fang-Yen, S. F. Fulghum, K. Badizadegan, R. R. Dasari, and M. S. Feld, “Assessing epithelial cell nuclear morphology by using azimuthal light scattering spectroscopy,” Opt. Lett. 31(21), 3119–3121 (2006).
A. Brunsting and P. F. Mullaney, “Differential Light Scattering from Spherical Mammalian Cells,” Biophys. J. 14(6), 439–453 (1974).
R. M. P. Doornbos, M. Schaeffer, A. G. Hoekstra, P. M. A. Sloot, B. G. deGrooth, and J. Greve, “Elastic light-scattering measurements of single biological cells in an optical trap,” Appl. Opt. 35(4), 729–734 (1996).
V. P. Maltsev, “Scanning flow cytometry for individual particle analysis,” Rev. Sci. Instrum. 71(1), 243–255 (2000).
V. P. Maltsev, and K. A. Semyanov, Characterisation of Bio-Particles from Light Scattering (VSP, Utrecht, 2004).
A. E. Zharinov, P. A. Tarasov, A. N. Shvalov, K. A. Semyanov, D. R. van Bockstaele, and V. P. Maltsev, “A study of light scattering of mononuclear blood cells with scanning flow cytometry,” J. Quant. Spectrosc. Radiat. Transf. 102(1), 121–128 (2006).
W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasari, and M. S. Feld, “Tomographic phase microscopy,” Nat. Methods 4(9), 717–719 (2007).
W. Choi, C. C. Yu, C. Fang-Yen, K. Badizadegan, R. R. Dasari, and M. S. Feld, “Field-based angle-resolved light-scattering study of single live cells,” Opt. Lett. 33(14), 1596–1598 (2008).
Y. Sung, W. Choi, C. Fang-Yen, K. Badizadegan, R. R. Dasari, and M. S. Feld, “Optical diffraction tomography for high resolution live cell imaging,” Opt. Express 17(1), 266–277 (2009).
A. J. Devaney, “Inverse-scattering theory within the Rytov approximation,” Opt. Lett. 6(8), 374–376 (1981).
E. Wolf, “Three-dimensional structure determination of semi-transparent objects from holographic data,” Opt. Commun. 1(4), 153–156 (1969).
H. Hulst, Light scattering by small particles (Dover Publications, New York, 1981).
C. L. Curl, C. J. Bellair, T. Harris, B. E. Allman, P. J. Harris, A. G. Stewart, A. Roberts, K. A. Nugent, and L. M. D. Delbridge, “Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy,” Cytometry A 65A(1), 88–92 (2005).
K. F. A. Ross, Phase contrast and interference microscopy for cell biologists (Edward Arnold Ltd, London, 1967).
Fig. 1 Cross-section of 3D refractive index tomograms and 2D angular scattering maps: (a) Original tomogram section of HT29 cell and (b) Tomogram section with nucleolus replaced with random nuclear index values. (c) Tomogram of the cell nucleus. The color bar indicates refractive index at wavelength of 633 nm. Scale bars indicate 5 μm. (d) Angular scattering intensity of the original cell tomogram. (e) Scattering intensity of the nucleolus. (f) Angular scattering of the nucleus. The color bar indicates intensity in logarithm base 10 with arbitrary units.
Fig. 2 Fitting the angular scattering distribution of nucleus with Mie theory: (a) The tomogram section of the nucleus is indicated with major axis (blue) and minor axis (red). (b) Angular scattering distribution along major axis (blue) and Mie theory fit (black). (c) Angular scattering distribution along major axis (red) and Mie theory fit (black). (d) Tomogram section of homogenized nuclear tomogram. (e) Scattering along major axis (blue) and Mie theory fit (black). (f) Scattering along minor axis (red) and Mie theory fit (black). The color bars in (a) and (d) indicate refractive index at the wavelength of 633 nm.
Fig. 3 Comparison of relative scattering strengths among the whole cell, the entire intracellular organelles, and the nucleus: (a) Tomogram of the HT29 cell in culture medium. (b) Tomogram of HT29 cell with index in the media matched to an average index of the whole cell. (c) Nuclear tomogram surrounded by the average index of the cytoplasm. Scale bars indicate 5 μm. (d) Angular scattering spectrum from the whole cell in the culture medium (blue), index-matched cell (green) and nucleus (red).

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