Infoscience

Thesis

Exploring dynamic organellar shape using live-cell fluorescence super-resolution microscopy

Eukaryotic cells contain membrane-bound organelles, that perform specialized tasks, which control cellular fate. Organelles are dynamic and contain nanometre-scale, ultrastructural features; in fact, their shape and function are interconnected. Thus, there is a need to image with high spatial and temporal resolution while minimizing perturbations to the cell. Several fluorescence-based super-resolution (SR) techniques exist, including single molecule localization microscopy (SMLM), which offers unsurpassed spatial resolution. However, this technique is limited by factors that prevent non-perturbative, fast, high resolution, quantitative imaging of biological processes. In particular, live-cell SMLM lacks flexible, robust labeling strategies; it requires high laser irradiances and toxic chemical buffers; and 3D data is often misrepresented due to optical distortions that are apparent at such high resolutions. Here, we aim to eliminate obstacles to live SMLM. Firstly, we screen site-specific dyes that are designed to target organelles. We identify dyes with excellent photophysical properties for live SMLM. Unlike many other probes for SMLM, these work well in non-toxic buffers. Secondly, we transform rhodamine dyes into their non-fluorescent leuco-rhodamine form. This transformation is reversible and occurs spontaneously through oxidation in situ. Labelling with this leuco-rhodamine dye increases single-molecule density, which is advantageous for SMLM since higher molecular densities yield improved spatial and temporal resolutions. Furthermore, this strategy does not require buffers and performs best at relatively low laser irradiances. Thirdly, we identify a distortion present in three-dimensional (3D) SMLM, which warps structures imaged. We characterize this prevalent distortion, termed wobble, on four 3D SMLM systems and identify its source. We computationally eliminate wobble and show that live-cell, 3D structures are accurately represented post-correction. Using this correction, we also develop a strategy to register dual-color, 3D SMLM data. Finally, we apply live-cell SMLM to study mitochondrial fission. Mitochondria are dynamic and frequently undergo fusion and fission to maintain their function and structure. The dynamin related protein, Drp1 in mammals, is required for mitochondrial fission and is known to mediate this process via a constriction force. The endoplasmic reticulum and actin are also reported to play a role in mitochondrial constriction and ultimately, fission. However, a model for the final stages of fission post constriction, is still missing. With this ultimate aim, we perform live SMLM of many mitochondrial fission events. We identify a decrease in viability with our imaging conditions and thus validate our findings with an alternative, more live-cell compatible, SR technique: structured illumination microscopy (SIM). Our preliminary results with live SMLM and SIM reveal that while most constrictions proceed to fission, 5% of these events relax back to their unconstricted shape. Measurements with SMLM reveal that minimum diameters for both scission events and those that reversed were 80 nm. Also, mitochondria, which achieve a higher negative envelope curvature, are more likely to divide. By examining Drp1 location, we find that this protein is consistently on one mitochondrial end post-scission. These preliminary results are discussed in the context of the final stages of mitochondrial fission.

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