Angularly resolved, finely sampled elastic scattering measurements of single cells: requirements for robust organelle size extractions

Abstract. Angularly resolved elastic light scattering is an established technique for probing the average size of organelles in biological tissue and cellular ensembles. Focusing of the incident light to illuminate no more than one cell at a time restricts the minimum forward-scattering angle θmin that can be detected. Series of simulated single-cell angular-scattering patterns have been generated to explore how size estimates vary as a function of θmin. At a setting of θmin  =  20  deg, the size estimates hop unstably between multiple minima in the solution space as simulated noise (mimicking experimentally observed levels) is varied. As θmin is reduced from 20 deg to 10 deg, the instability vanishes, and the variance of estimates near the correct answer also decreases. The simulations thus suggest that robust Mie theory fits to single-cell scattering at 785 nm excitation require measurements down to at least 15 deg. Notably, no such instability was observed at θmin  =  20  deg for narrow bead distributions. Accurate sizing of traditional calibration beads is, therefore, insufficient proof that an angular-scattering system is capable of robust analysis of single cells. Experimental support for the simulation results is also presented using measurements on cells fixed with formaldehyde.

[1]  Thomas H Foster,et al.  Index-of-refraction-dependent subcellular light scattering observed with organelle-specific dyes. , 2007, Journal of biomedical optics.

[2]  J P Freyer,et al.  Polarized angular dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures. , 2002, Journal of biomedical optics.

[3]  Yongjin Sung,et al.  Assessing light scattering of intracellular organelles in single intact living cells. , 2009, Optics express.

[4]  Judith R. Mourant,et al.  Light scattering from cells: the contribution of the nucleus and the effects of proliferative status , 2000, BiOS.

[5]  Vadim Backman,et al.  Simultaneous measurement of angular and spectral properties of light scattering for characterization of tissue microarchitecture and its alteration in early precancer , 2003 .

[6]  T. Foster,et al.  Mie theory interpretations of light scattering from intact cells. , 2005, Optics letters.

[7]  Adam Wax,et al.  Application of the T-matrix method to determine the structure of spheroidal cell nuclei with angle-resolved light scattering. , 2008, Optics letters.

[8]  Michael S. Feld,et al.  Polarized light scattering spectroscopy for quantitative measurement of epithelial cellular structures in situ , 1999 .

[9]  Gérard Gréhan,et al.  Localized interpretation to compute all the coefficients gnm in the generalized Lorenz–Mie theory , 1990 .

[10]  T. Foster,et al.  Microscope enabling multimodality imaging, angle-resolved scattering, and scattering spectroscopy. , 2007, Optics letters.

[11]  Thomas B. A. Senior,et al.  Absorption and Scattering by Small Particles; Structure of the Internal and Near Fields. , 1984 .

[12]  Gérard Gréhan,et al.  Scattering of a Gaussian beam by a Mie scatter center using a Bromwich formalism , 1985 .

[13]  Angela A. Eick,et al.  Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics. , 1998, Applied optics.

[14]  Nicole J. Moore,et al.  Closed form formula for Mie scattering of nonparaxial analogues of Gaussian beams. , 2008, Optics express.

[15]  G Gouesbet,et al.  Scattering of laser beams by Mie scatter centers: numerical results using a localized approximation. , 1986, Applied optics.

[16]  Zhuo Wang,et al.  Fourier transform light scattering of inhomogeneous and dynamic structures. , 2008, Physical review letters.

[17]  Kamran Badizadegan,et al.  Field-based angle-resolved light-scattering study of single live cells. , 2008, Optics letters.

[18]  Jing-Yi Zheng,et al.  Alterations in the characteristic size distributions of subcellular scatterers at the onset of apoptosis: effect of Bcl-xL and Bax/Bak. , 2010, Journal of biomedical optics.

[19]  Thomas H Foster,et al.  Light scattering from intact cells reports oxidative-stress-induced mitochondrial swelling. , 2005, Biophysical journal.

[20]  A. Wax,et al.  Determining nuclear morphology using an improved angle-resolved low coherence interferometry system. , 2003, Optics express.

[21]  Andrew J Berger,et al.  Validation of an integrated Raman- and angular-scattering microscopy system on heterogeneous bead mixtures and single human immune cells. , 2009, Applied optics.

[22]  Michael B. Wallace,et al.  Observation of periodic fine structure in reflectance from biological tissue: A new technique for measuring nuclear size distribution , 1998 .

[23]  G. Mie.  Beiträge zur Optik trüber Medien, speziell kolloidaler Metallösungen , 1908 .

[24]  Wilsaan M. Joiner,et al.  BCL-xL-dependent light scattering by apoptotic cells. , 2004, Biophysical journal.

[25]  G. Gouesbet,et al.  The order of approximation in a theory of the scattering of a Gaussian beam by a Mie scatter center , 1985 .

[26]  Denis Cousineau,et al.  Standard errors: A review and evaluation of standard error estimators using Monte Carlo simulations , 2014 .