Mitochondrial dynamics such as fission, fusion and movement can reveal the physiological status of neurons and, most importantly, serve as diagnostic measures for several neurodegenerative diseases. Traditionally fluorescent probes have been used to track mitochondrial dynamics in neurons. However, neurons show low transfection efficiency, presenting challenges for the use of genetically-encoded fluorescent markers. Alternatively, synthetic fluorescent dyes are shown to hinder mitochondrial motility. In addition, all types of fluorescent probes are subject to photo-bleaching which precludes imaging for longer periods of time. To circumvent these issues, we propose a light-scattering based label-free technique called Optical Scatter Imaging (OSI) that is sensitive to changes in morphology. In this work, we employ a previously reported label-free parameter to probe the change in the size of organelles such as mitochondria as they undergo fusion or fission in neurons. In addition, we present a technique to track organelle motion using kymographs obtained from the label-free images and compare them with those obtained from fluorescent images. We demonstrate that the label-free kymograph can track organelles such as mitochondria even after the sample is photo-bleached.
Imaging without fluorescent protein labels or dyes presents significant advantages for studying living cells without confounding staining artifacts and with minimal sample preparation. Here, we combine label-free optical scatter imaging with digital segmentation and processing to create dynamic subcellular masks, which highlight significantly scattering objects within the cells’ cytoplasms. The technique is tested by quantifying organelle morphology and redistribution during cell injury induced by calcium overload. Objects within the subcellular mask are first analyzed individually. We show that the objects’ aspect ratio and degree of orientation (“orientedness”) decrease in response to calcium overload, while they remain unchanged in untreated control cells. These changes are concurrent with mitochondrial fission and rounding observed by fluorescence, and are consistent with our previously published data demonstrating scattering changes associated with mitochondrial rounding during calcium injury. In addition, we show that the magnitude of the textural features associated with the spatial distribution of the masked objects’ orientedness values, changes by more than 30% in the calcium-treated cells compared with no change or changes of less than 10% in untreated controls, reflecting dynamic changes in the overall spatial distribution and arrangement of subcellular scatterers in response to injury. Taken together, our results suggest that our method successfully provides label-free morphological signatures associated with cellular injury. Thus, we propose that dynamically segmenting and analyzing the morphology and organizational patterns of subcellular scatterers as a function of time can be utilized to quantify changes in a given cellular condition or state.
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.