Fluorescence imaging of cellular organelles and membrane dynamics

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Wijesooriya, Chamari
Major Professor
Emily A. Smith
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Fluorescence microscopy is a versatile technique for studying biological structures in their native environment. The live cell imaging ability of this technique enables the study of dynamic processes in biological systems. Fluorophores play an important role in fluorescence-based cellular imaging. Development of cell permeable, non-toxic, small organic molecules with specific targeting ability is advantageous for imaging of live cellular organelles. Two coumarin-based compounds were developed to selectively target the endoplasmic reticulum (ER), which is the largest organelle in most mammalian cell types. Unlike the commercially available ER targeting probes, the coumarin-based compounds can be used to image the ER in both live and fixed cells. The simple synthetic procedure, bright emission in the blue region, narrow emission profile, low toxicity, and the specificity contribute to the utility of these probes for imaging the dynamic events of the ER.

The spatial resolution the traditional optical microscopy is limited by the diffraction of light. Subcellular organelles are in the nanometer size range, and the majority of the biological phenomena happen in the nanoscale. Therefore traditional light microscopic techniques have limited capability of studying these systems. Super-resolution microscopic techniques that were developed in the past decade to overcome this issue. Single molecule localization microscopy (SMLM) is based on the activation and excitation of subsets of fluorophores, followed by their localization with nanometer spatial resolution to generate a super-resolution image. Traditional SMLM fluorophores have the drawbacks of requiring two high power lasers including lasers emitting in the ultra-violet range. Also, the requirement of harsh chemicals in the imaging medium limits the ability to image live cells. A BODIPY-based photoactivatable probe is developed as a promising probe for SMLM. A single low-power visible laser is utilized without requiring harsh conditions in the imaging medium to make this probe exceptionally useful for imaging in biological systems. The probe can be simply linked to nucleophile-containing targeting groups to image specific cellular components. Super-resolution images of in vitro and in vivo microtubules are generated using paclitaxel attached probe.

Membrane receptors are one of the major components that contribute to the control of cellular function. Interactions of receptor proteins with extracellular ligands, other membrane constituents, and intracellular components result in the activation of intracellular signaling mechanisms. These dynamic interactions are governed by the lateral diffusion of membrane components. RAGE is a multi-ligand receptor responsible for various pathological diseases. Amyloid beta 1-40 and 1-42 peptides react with RAGE protein to stimulate neuronal dysfunction and are also responsible for Alzheimer's disease. The effects of these peptides on RAGE diffusion is not yet known. The fluorescence-based single particle tracking method is used to measure the effect of amyloid beta ligands for the lateral diffusion of the receptor for advanced glycation endproducts (RAGE) at the single receptor level. Both of the peptides altered the RAGE membrane diffusion, but to a different extent. Activation of the p38 MAPK pathway is observed for the treatment of RAGE with amyloid beta 1-42 ligand. The effect of different oligomeric forms of these two peptides on the RAGE diffusion and signaling is further studied.

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Sun Dec 01 00:00:00 UTC 2019