Assessing the contribution of cell body and intracellular organelles to the backward light scattering

Abstract

We report a method of assessing the contribution of whole cell body and its nucleus to the clinically most relevant backward light scattering. We first construct an experimental system that can measure forward scattering and use the system to precisely extract the optical properties of a specimen such as the refractive index contrast, size distribution, and their density. A system that can simultaneously detect the backscattered light is installed to collect the backscattering for the same specimen. By comparing the measured backscattering spectrum with that estimated from the parameters determined by the forward scattering experiment, the contribution of cell body and nucleus to the backward light scattering is quantitatively assessed. For the HeLa cells in suspension, we found that the cell body contributes less than 10% and cell nucleus on the order of 0.1% to the total backscattering signal. Quantitative determination of the origin of backscattered light may help design a system that aims for detecting particular structure of biological tissues.

Fig. 1

Polar plots of differential scattering cross-section σ as a function of scattering angles θ and φ (Mie simulation). Color bar is the magnitude of the differential cross-section in μm²/sr. Origin of the maps is either 0° (forward scattering) or 180° (backscattering). Top row for 5 μm-size particle. Bottom row for 50 nm-size particle. (a,b) Backscattering parallel cross-section. (c,d) Backscattering perpendicular cross-section. (e,f) Forward scattering cross-section.

Fig. 2

Light scattering instrument. System components: P1, P2 incident beam polarizers, BS-beam splitter, A – analyzer, FP – Fourier plane, F-focal distance of Fourier Lens, IL1 and IL2 – imaging lenses with focal distances of a and b. Traces of the incident light are shown in green. Brown and light brown traces belong to scattered light at 0°, 180° and at θ degrees away from them.

Fig. 3

(a) Phase contrast image of HeLa cells at 40x magnification. (b) Light scattering measurements normalized in wavelength λ and angle θ. Color bar is in 1/R99 units.

Fig. 4

Results of fitting experimental data to Mie theory. Scattering measurements and Mie theory are mean-centered. (a) Wavelength spectrum measurements (blue) at fixed scattering angles with fits to Mie theory (red). (b) Angular spectrum measurements (blue) at fixed wavelengths with fits to Mie theory (red).

Fig. 5

Phase contrast image of HeLa cells in regular culture media (a) and in the media containing BSA (b). Measured forward light scattering distribution in culture media (c) and in BSA solution (d). Angular light scattering distribution for BSA and no-BSA samples from the measurement (e) and Mie theory (f).

Fig. 6

(a) Measured backscattering cross-section of the HeLa cells in suspension. (b) Calculated backscattering cross-section for the whole cell body. Both intensities are in 1/R99 units. (c) Comparison of the cross-sections at θ = 178°.