Optical diffraction tomography for assessing single cell models in angular light scattering.

Angularly resolved light scattering (ALS) has become a useful tool for assessing the size and refractive index of biological scatterers at cellular and organelle length scales. Sizing organelle populations with ALS relies on Mie scattering theory models, which require significant assumptions about the object, including spherical scatterers and a homogeneous medium. These assumptions may incur greater error at the single cell level, where there are fewer scatterers to be averaged over. We investigate the validity of these assumptions using 3D refractive index (RI) tomograms measured via optical diffraction tomography (ODT). We compute the angular scattering on digitally manipulated tomograms with increasingly strong model assumptions, including RI-matched immersion media, homogeneous cytosol, and spherical organelles. We also compare the tomogram-computed angular scattering to experimental measurements of angular scattering from the same cells to ensure that the ODT-based approach accurately models angular scattering. We show that enforced RI-matching with the immersion medium and a homogeneous cytosol significantly affects the angular scattering intensity shape, suggesting that these assumptions can reduce the accuracy of size distribution estimates.

Three-dimensional angular scattering simulations inform analysis of scattering from single cells.

Significance: Organelle sizes, which are indicative of cellular status, have implications for drug development and immunology research. At the single cell level, such information could be used to study the heterogeneity of cell response to drugs or pathogens. Aim: Angularly resolved elastic light scattering is known to be sensitive to changes in organelle size distribution. We developed a Mie theory-based simulation of angular scattering from single cells to quantify the effects of noise on scattering and size estimates. Approach: We simulated randomly sampled organelle sizes (drawn from a log normal distribution), interference between different organelles' scattering, and detector noise. We quantified each noise source's effect upon the estimated mean and standard deviation of organelle size distributions. Results: The results demonstrate that signal-to-noise ratio in the angular scattering increased with the number of scatterers, cell area, and exposure time and decreased with the size distribution width. The error in estimating the mean of the size distributions remained below 5% for nearly all experimental parameters tested, but the widest size distribution tested (standard deviation of 600 nm) reached 20%. Conclusions: The simulator revealed that sparse sampling of a broad size distribution can dominate the mismatch between actual and predicted size parameters. Alternative estimation strategies could reduce the discrepancy.

Matching an immersion medium's refractive index to a cell's cytosol isolates organelle scattering.

Angularly-resolved light scattering has been proven to be an early detector of subtle changes in organelle size due to its sensitivity to scatterer size and refractive index contrast. However, for cells immersed in media with a refractive index close to 1.33, the cell itself acts as a larger scatterer and contributes its own angular signature. This whole-cell scattering, highly dependent on the cell's shape and size, is challenging to distinguish from the desired organelle scattering signal. This degrades the accuracy with which organelle size information can be extracted from the angular scattering. To mitigate this effect, we manipulate the refractive index of the immersion medium by mixing it with a water-soluble, biocompatible, high-refractive-index liquid. This approach physically reduces the amount of whole-cell scattering by minimizing the refractive index contrast between the cytosol and the modified medium. We demonstrate this technique on live cells adherent on a coverslip, using Fourier transform light scattering to compute the angular scattering from complex field images. We show that scattering from the cell: media refractive index contrast contributes significant scattering at angles up to twenty degrees and that refractive index-matching reduces such low-angle scatter by factors of up to 4.5. This result indicates the potential of refractive index-matching for improving the estimates of organelle size distributions in single cells.

Phase-sensitive, angle-resolved light-scattering microscopy of single cells.

We report what is to our knowledge the first use of Fourier phase microscopy (FPM) to estimate diameters of individual single-micrometer beads and to classify cells based upon changes in scatterer size distribution. FPM, a quantitative phase imaging (QPI) method, combines the planar illumination typically used in off-axis QPI (ideal for Mie theory analysis) with the common-path geometry typically used in on-axis QPI (ideal for optimizing angular scattering range). Low-spatial-frequency imaging artifacts inherent to FPM have negligible impact upon these angular-domain applications. The system is simple to align and stable, and requires no external reference beam. Angular scattering patterns obtained from single 1 µm polystyrene beads in glycerol (Δn=0.11) display unprecedented fidelity to Mie theory, produce diameter estimates consistent with the manufacturer's specifications, and offer precision on the scale of tens of nanometers. Measurements of macrophages at different stages of antibody-dependent cellular phagocytosis demonstrate the ability to detect changes in a cell's scattering caused by the presence of phagocytosed material within.