Is the nuclear refractive index lower than cytoplasm? Validation of phase measurements and implications for light scattering technologies - PubMed (original) (raw)

Is the nuclear refractive index lower than cytoplasm? Validation of phase measurements and implications for light scattering technologies

Zachary A Steelman et al. J Biophotonics. 2017 Dec.

Abstract

The refractive index (RI) of biological materials is a fundamental parameter for the optical characterization of living systems. Numerous light scattering technologies are grounded in a quantitative knowledge of the refractive index at cellular and subcellular scales. Recent work in quantitative phase microscopy (QPM) has called into question the widely held assumption that the index of the cell nucleus is greater than that of the cytoplasm, a result which disagrees with much of the current literature. In this work, we critically examine the measurement of the nuclear and whole-cell refractive index using QPM, validating that nuclear refractive index is lower than that of cytoplasm in four diverse cell lines and their corresponding isolated nuclei. We further examine Mie scattering and phase-wrapping as potential sources of error in these measurements, finding they have minimal impact. Finally, we use simulation to examine the effects of incorrect RI assumptions on nuclear morphology measurements using angle-resolved scattering information. Despite an erroneous assumption of the nuclear refractive index, accurate measurement of nuclear morphology was maintained, suggesting that light scattering modalities remain effective.

Keywords: Mie theory; light scattering; microscopy; nucleus; phase imaging; refractive index.

© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figures

Figure 1

Figure 1

Refractive index of whole cells and nuclei for each chosen cell line at each chosen wavelength. Chromatic dispersion of cellular material is generally 10−5 nm−1 or less. The number of samples analyzed was Ncell = 16, 17, 12, 25 and Nnuc = 10, 15, 15, 19 for MCF-7, A549, BEAS-2B, and HVE whole cells and nuclei, respectively. A nuclear refractive index which is lower than that of cytoplasm appears to be an accurate characterization of the cellular substructure.

Figure 2

Figure 2

(Top Left) Standard QPM fails to properly reconstruct the phase profile of a polymer microsphere for λ ≤ 660 nm. (Bottom left) Synthetic wavelength imaging with two wavelengths (Two-λ QPM) reveals hidden phase wraps which were not properly reconstructed by Goldstein’s algorithm. (Right) Two-λ QPM was applied to all cells and nuclei in the study (Pictured: MCF-7), though no additional 2π phase delays were uncovered in any cell or nucleus. Phase wrapping does not cause artificial reduction of the RI measurement of nuclei using QPM.

Figure 3

Figure 3

Simulated fitting of angular scattering spectra from low-index nuclei, using a high-index lookup table. (Top) A sample angular spectrum, m < 1, along with its best fit, _m_ > 1. In this case, the nuclear diameter is correctly determined to be 12.7 μm. (Middle left) All simulations on the parameter space. Size accuracy is limited for individual measurements. (Middle Right) Average fit for groups of N=7 spectra with a size standard deviation of 10% from the test diameter. (Bottom) Bland-Altman plot for the size-averaged fits. Half of the error comes from a systematic offset of 0.691 μm, which does not affect diagnostic accuracy. Ignoring the offset, the remaining error has standard deviation of 0.689 μm. Red lines indicate 95% limits of agreement, or ±1.96 times the standard deviation of the error. All simulations were performed at 792.1 nm, the measured center wavelength of a benchtop a/LCI system in our lab.

Figure 4

Figure 4

(Left) Distribution of mean nuclear diameters for simulated healthy and dysplastic cervical tissues, N=100 each. CIN II/III represents expected nuclear morphology for a high-grade squamous intraepithelial lesion (HSIL). (Middle) Individual size measurements of the input diameters on the left using the m > 1 library. (Right) Sample averaging of N=7 improves diagnostic accuracy. The measured healthy distribution is indistinguishable from the input, p > 0.1. The dysplastic population is systematically shifted upwards by 1 μm, but indistinguishable from the input (p > 0.5) when this is corrected. A suggested decision line at 11 μm is included for illustrative purposes.

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