Label-free Fourier filtered dark-field imaging to quantify subcellular dynamics

Light scattering by subcellular organelles and interfaces such as membranes can be utilized for quantitative measurement of cellular and tissue states. The structural information of the organelles can be inferred from light-scattering by analyzing the signal at a conjugate Fourier plane of a dark-field imaging system. Via implementation of Gabor filters on the Fourier plane, we can selectively allow only certain angles of scattering to pass during imaging. These scatter angles are related to the spatial frequencies of the scattering source. So in effect, the Gabor filters can be tuned to probe objects of certain size/shape and orientation. Based on this property, we had previously reported a morphometric parameter called Orientedness that can detect change in mitochondrial orientation during apoptosis. In this work, we present a subcellular segmentation technique to track Orientedness values over time in individual subcellular structures. This is achieved through a dynamic mask that changes its shape according to the shape of the organelles while they undergo chemically-induced morphological changes. The mask is generated from the Gabor-filtered images of the cell. We also propose a modification of the original Orientedness calculation using local-energy information. We demonstrate our method by tracking the morphology of subcellular structures within cells which have been overloaded with calcium. Our results show changes in the morphology of the subcellular structure in calcium-treated cells but not in the untreated control. Moreover a decrease in the aspect ratio and orientedness of the analyzed structures correlates with the onset of mitochondrial rounding and fission as seen in fluorescence. Together, our results suggest that light scattering based labelfree analysis of organelle structures may be used to track subcellular activity over time.