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theses

Live-cell imaging has become increasingly important to the scientific community as researchers aim to understand the morphology and dynamics of the subcellular structures. Specifically, mitochondrial morphology under healthy and pathologic conditions is an emerging area of interest, with applications in neuroprotective drug discovery amongst many. Fluorescent dyes act as the current biological gold standard for visualizing organelles in live-cell imaging, yet they have been shown to cause adverse effects on subcellular functions, prompting the development of alternate, “label-free”, methods of imaging. By employing Optical Scatter Imaging (OSI), which is based on optical Fourier filtering where scattered light is filtered by the hardware using size and orientation sensitive Gabor filters, images can be acquired label-free and processed to extract morphometric data. As groundwork for developing a label-free method for probing mitochondria, we first demonstrated that optical scatter imaging with principal component analysis (PCA) can classify apoptosis-competent Bax/Bak-expressing, and apoptosis resistant Bax/Bak-null immortalized baby mouse kidney (iBMK) cells without the use of fluorescent labels. Furthermore, in Bovine Aortic Endothelial Cells (BAEC), a different cell type, we revealed that label-free Optical Scatter Imaging (OSI) can be combined with digital segmentation to create dynamic subcellular masks. These masks can be used to extract the “orientedness” of organelles, which corresponds to an organelle’s degree of orientation, allowing the quantification of organelle morphology during cell injury induced by calcium overload. In order to optimize this label free imaging and processing method for neuronal mitochondria, we applied a size-encoding parameter, “Smax”. All in all, we have developed different approaches to detect and quantify morphological changes associated with mitochondria and other organelles in three different cell-types and we have compared the results with a fluorescent data. This ultimately implies that our label-free imaging technique can be modified for use in many cell types, enabling numerous possible research applications in organelle visualization.

ETD_9211

Ph.D.

Includes bibliographical references

by Mohammad Naser

doi:10.7282/t3-px37-sf74

ETD doctoral

The author owns the copyright to this work.