Three-dimensional computation of light scattering by multiple biological cells
This work presents an investigation into the optical scattering of heterogeneous cells with an application to two-photon imaging, optical scattering measurements and STED imaging. Using the finite difference time-domain (FDTD) method, the full-wave scattering by many cells containing multiple organelles with varying indices of refraction is computed. These simulations were previously limited to single cells for reasons of computational cost.
A superposition approximation that uses the coherent linear superposition of FDTD-determined farfield scattering patterns of small numbers of cells to estimate the scattering from a larger tissue was developed and investigated. It was found that for the approximation to be accurate, the scattering sub-problems must at minimum extend along the incident field propagation axis for the full depth of the tissue, preserving the scattering that takes place in the direction of propagation.
The FDTD method was used to study the scattering effects of multiple inhomogeneous cells on the propagation of a focused Gaussian beam with an application to two-photon imaging. It was found that scattering is mostly responsible for the reduction in two-photon fluorescence signal as depth is increased. It was also determined that for the chosen beam parameters and the cell and organelle configurations used, the nuclei are the dominant scatterers.
FDTD was also utilized in an investigation of cellular scattering effects on the propagation of a common depletion beam used in STED microscopy and how scattering impacts the image obtained with a STED microscope. An axial doughnut beam was formulated and implemented in FDTD simulations, along with a corresponding focused Gaussian beam to simulate a fluorescence excitation beam. It was determined that the depletion beam will maintain a well-defined axial null in spite of scattering, although scattering will reduce the resulting fluorescence signal with focal depth.