FLUCTUATIONS IN SINGLE-CELL ORGANELLE SIZE ESTIMATES FROM ANGULAR SCATTERING MEASUREMENTS

摘要

Angularly resolved elastic scattering microscopy enables the size distribution of cellular organelles to be estimated non-invasively. By comparing the angular distribution of scattered light to Mie theory models, our group is working towards obtaining quantitative estimates of mean organelle size within single cells. Recent work, for example, established that the lower end of the angular range plays a critical role. Both simulations and measurements demonstrate that lowering the minimum measured angle from 20 degrees to below 15 degrees is essential for obtaining stable size estimates from a single cell. Even with the lower angular range included, however, there are noticeable differences in how much size predictions fluctuate in time for live versus fixed cells, as indicated in the second figure below. Potential mechanisms for these fluctuations are being investigated. One mechanism could be that organelles are moving into and out of the beam over time. In previous work, our beam has overlapped only a portion of the cell's cross-section, allowing this effect to take place. Increasing the spot diameter to overlap the entire cell will remove this degree of freedom. Other potential causes of the greater fluctuation in live-cell measurements include (a) time-varying speckle caused by organelles moving relative to one another and (b) true size changes in the overall organelle population overtime. Speckle mitigation techniques can be explored in simulations and experimentally with interferometric methods. Size changes in specific organelles such as mitochondria or lysosomes can be induced by various physical and chemical effects. Investigation of these factors will determine the smallest size changes that can be detected in a single cell's organelle population.