Thesis (Ph. D.)--University of Rochester. Institute of Optics, 2014.
The size of organelles can indicate many things about the health or behavior
of cells. Elastic light scattering provides a promising method to non-invasively
measure organelle sizes in living cells. Angle-resolved light scattering of multi-
cell suspensions or tissues has been used to diagnose dangerous pre-cancerous
conditions and track changes in sub-cellular structure during apoptosis.
Angular scattering measurements of single cells would allow researchers to ex-
amine differences in structure and behavior among individual cells. Unfortunately,
single cell scattering measurements are more difficult because the small illuminated
region leads to larger speckle grains. This speckle causes organelle size estimates
to be inaccurate and unstable.
This thesis presents a holographic angular domain elastic scattering (HADES)
system that interferometrically measures the full complex field of scattered light. It
also describes multiple methods of using this complex field to overcome the effects
of speckle and obtain scatterer-size estimates. Two methods are presented that are
appropriate for analyzing ensembles of discrete scatterers (e.g., micro-beads). Sub-
cellular structure is more continuous, so another method is used to reduce speckle
in HADES measurements of cells. Digital spatial coherence reduction (DiSCoRd)
decreases the correlation between scatterers using phase masks and synthetically
expands the illuminated region. This in silico processing effectively reduces speckle
even when the scatterer locations are less defined (e.g., in biological cells).
The accuracy of the HADES technique is confirmed by measurements of groups
of stationary polystyrene beads. Intensity-based scattering measurements of these
samples are dominated by speckle, whereas sizes extracted by HADES measure-
ments are accurate to less than 27 nm.
HADES measurements of single cells demonstrate the precision and stabil-
ity of time-lapsed interferometric measurements. Organelle size estimates from
untreated cells vary by less than 57 nm over 2 hours. Experiments measuring
calcium-induced mitochondrial swelling demonstrate the potential of the HADES
technique to measure changes in organelle sizes over time.