Angularly resolved elastic scattering microscopy enables the size distribution of cellular organelles to be estimated non-invasively. By comparing the angular distribution of light scattered from a cell to Mie theory models, our group is working towards obtaining quantitative estimates of mean organelle size within single cells and tracking them over time. These estimates can then be used to assess the health of the cell. We are now testing our system’s performance in measuring induced size changes in HeLa cells undergoing apoptosis, or programmed cell death. Apoptosis is induced in these cells by exposing them to a Fas ligand. Because the response of the cell occurs on the scale of a few hours, we can take multiple measurements of many cells individually before they show changes that would be visible with traditional microscopy. Additionally, we take measurements prior to exposing cells to the ligand to establish the baseline fluctuations in the organelle size estimates. Our goal is to find optical signatures that characterize the cellular progression prior to the final, most extreme stages of the apoptotic process. We would then be able to look at the behavior of multiple cells and predict when each cell will die.
The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.