Mitochondria are the power plant of most non-green eukaryotic cells. In order to understand mitochondrial functions and their regulation, knowledge of the spatiotemporal organization of their proteins is important. Mitochondrial membrane proteins can diffuse within membranes. They are involved in diverse functions e.g. protein import, cell respiration, metabolism, metabolite transport, fusion, fission or formation of the mitochondrial architecture. Furthermore, mitochondria compose of different subcompartments with different tasks. Especially, the inner mitochondrial membrane (IM), where the oxidative phosphorylation (OXPHOS) takes places, has a complex architecture with cristae extending into the matrix. The present work revealed the restricted localization of some mitochondrial proteins to specific membrane sections and linked it to their function or bioenergetic circumstances in the living cell. 
Single particle tracking (SPT) techniques like tracking and localization microscopy (TALM) allow to localize proteins with a precision below 20 nm. Additionally, tracking single proteins provides information about their mobility, dynamic and their spatiotemporal organization. TALM uses proteins, which were genetically tagged either with the HaloTag® (HaloTag) or the fSnapTag® (fSnapTag). These tags can be orthogonally and posttranslationally stained with specific and self-marking dyes. If the dyes are conjugated to the respective substrate of the tag. Single molecule labeling of mitochondrial proteins was performed substoichiometrically using membrane permeable rhodamine dyes, either tetramethylrhodamine (TMR) or silicon rhodamine (SiR). TALM allowed to localize proteins in different mitochondrial subcompartments. The gained trajectories and trajectory maps of mitochondrial proteins revealed their spatiotemporal organization. In the case of IM proteins like F1FO ATP synthase (Complex V - CV) a restricted diffusion in the CM, which is part of the continuous IM, was determined. The unimpeded diffusion of mitochondrial proteins in the outer mitochondrial membrane (OM) was compared with the mobility of IM proteins. The diffusion of mitochondrial IM proteins was restricted by the IM architecture and their diffusion coefficients were lower. Furthermore, significant differences of different mitochondrial IM proteins were compared, showing different localizations in the IM often coupled to their function, accompanied by different spatiotemporal organization and diffusion coefficients. Furthermore, a distinction was made between diffusion of proteins in the inner boundary membrane (IBM) and proteins that preferentially diffuse in the cristae membrane (CM). Evaluating trajectory maps, the different subcompartments in the IM were revealed by trajectories and the trajectory directionality, allowing the identification of mitochondrial proteins, which mark these subcompartments.
The morphology of mitochondria / mitochondrial networks and their bioenergetic parameters are linked to the metabolic states of the cell. In this work, the connection of the spatiotemporal protein organization of CV and the IM architecture was uncovered on the micro- and nanoscopic level and linked to the metabolic state of the cell. It was determined that the spatiotemporal organization of the CV was altered, when CV was inhibited. In addition, the bioenergetic influence of cells on the spatiotemporal behavior of CV and the reorganization of the IM architecture was investigated by TALM and compared with results of electron microscopy images. It was shown that starvation of cells led to a loss of cristae and thus to an increased mobility and spatiotemporal reorganization of CV. Taken together, the results presented in this work showed that a correctly functioning and active CV helps to maintain the IM architecture and both, the spatiotemporal organization of CV and the IM architecture were coupled to the metabolic state.. 
In order to investigate putative protein-protein interactions by colocalization and co-locomotion studies on single molecule level, dual color SPT is needed. Therefore, posttranslational and substoichimetric labeling as performed in TALM was tested for its potential of protein-protein interaction studies of mitochondrial membrane proteins. Here, a genetically double tagged translocase of the outer membrane subunit-20 (Tom20) (Tom20:HaloTag:fSnapTag) acted as a positive control. It turned out that substoichimetric, posttranslational labeling of mitochondrial proteins was not suitable for protein-protein interaction studies on mitochondrial proteins, because it was restricted by the low labeling degrees needed for TALM. However, dual-color TALM still allowed to study effects of proteins influencing the IM architecture and to study their influence on the spatiotemporal organization of CV. The co-transfection of Mic10, as the central protein of the mitochondrial inner membrane organizing system / mitochondrial contact site complex / mitochondrial organizing structure (MINOS / MICOS / MitOS (MINOS/MICOS)), altered the regular and aligned organization of the cristae. This was measured by a changed spatiotemporal organization of the CV, such as the loss of the perpendicular oriented of CV subunit-γ (CV-SUγ) cristae trajectories. In contrast to this, co-transfection of CV subunit-e (CV-SUe), important for dimerization of CV, increased the number of cristae trajectories. 
Mitochondria are three-dimensional (3D) cell organelles. Consequently, subcompartments like the IBM and CM are a 3D space in which CV is localized and diffuses. Thus, the diffusion of mitochondrial proteins is underestimated by two-dimensional SPT e.g. lateral confined diffusion can result from mitochondrial proteins diffusing along the z-axis of the microscope. In order to reveal the 3D spatiotemporal organization of CV, the potential of TALM to be extended to a 3D-SPT technique was investigated. Therto a cylindrical lens was installed in the emission path of a total internal reflection fluorescence (TIRF) microscope. This leads to an astigmatically distorted point spread function (PSF) of the fluorescent single molecule signals. This distortion allowed the reconstruction of single molecule localizations of CV to a superresolved image of the IM, in living cells. In addition, 3D-TALM enabled to display the 3D architecture of the IM by 3D trajectories of CV. 3D-TALM was able to detect whether CV diffuses in the IBM or in the CM, and extended the information about its mobility in the CM that it takes place in a disc-like manner. In this way it could be shown that CV is mobile within the cristae in all directions. Finally, 3D-TALM revealed an altered IM architecture caused by the metabolic state of the cell. As performed in two-dimensional TALM, the cells were kept under starving conditions. Here the now tubular IM architecture was revealed by 3D-TALM. The reversed metabolic state under improved respiratory conditions unexpectedly led to a more diverse IM architecture. These ultrastructural changes were also revealed by electron microscopy. Consequently, 3D-TALM enabled the study of IM architecture by tracking CV under different metabolic conditions, allowing an ultrastructural analysis of mitochondria in living cells. In addition, 3D TALM provided the spatiotemporal organization of CV under different metabolic conditions, so that the diffusion coefficients of CV could be related to changes in IM architecture caused by the metabolic condition.

​My PhD thesis is an in-depth study of the magnetic properties of a series of different lanthanide-based endohedral metallofullerenes. They are sphere-like shape carbon molecules (fullerenes) with embedded magnetic lanthanide elements inside, suitable for spintronic and high dense-data storage applications. In this work, I studied two families of endohedral metallofullerenes (di-lanthanides and Dy-oxides) which showed great versatility in the magnetic behavior, depending on the type of the encapsulated cluster, and the size and shape of the carbon cage.:Magnetic studies on lanthanide dimetallofullerenes Gd2@C80(CH2Ph) and Gd2@C79N Tb2@C80(CH2Ph) and Tb2@C79N TbY@C80(CH2Ph) Ho2@C80(CH2Ph) Er2@C80(CH2Ph) Magnetic studies on Dy-oxide clusterfullerenes Dy2O@C72 Dy2O@C74 Dy2O@C82 (three isomers)

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In this work, we use optical techniques to provide insight into how various components within electrochemical cells can impart apparent heterogeneity to single gold nanoparticle electrodes. Optical methods are advantageous in comparison to traditional electrochemical techniques due to their high sensitivity and spatial resolution, allowing us to study the impact of heterogeneity with single nanoparticle and single molecule sensitivity. Throughout the course of this dissertation, two optical techniques are discussed in detail, dark-field microscopy, and single molecule fluorescence imaging. We first began by studying the impact of the substrate using dark-field microscopy to monitor the electrodissolution kinetics of gold nanoparticles on thin films of tin-doped indium oxide (ITO), which is a commonly used supporting electrode for correlated optical and electrochemical studies. We found that ITO from two different suppliers showed marked differences in the gold electrodissolution kinetics, with ITO from one of the suppliers even showing poor sample-to-sample reproducibility across substrates within the same lot number. These results showed that the supporting electrode cannot be ignored when performing single nanoparticle structure-function studies. In the second work, we analyzed the electrodissolution of gold nanoparticles on well-behaved ITO substrates to investigate heterogeneity in their electrodissolution kinetics. The rate constants associated with the electrodissolution of Au NPs were extracted by fitting the intensity-time traces to a first-order kinetic model. We found that a non-negligible population of Au NPs didn’t fit the predictive kinetics model leading us to further probe whether surface effects play a role in the electrodissolution process. Super-localization imaging was used to track the center position of the Au NPs as they electrodissolved revealing three distinct electrodissolution behaviors, and a mechanism for the electrodissolution of Au NPs was proposed. Furthermore, calcite-assisted localization and kinetics (CLocK) microscopy was used to visualize changes in anisotropy and provide information as to how the shape of the Au NP changes as it electrodissolves. Lastly, in our third work, we provide insight as to how heterogeneity from all the different components of a single nanoparticle electrochemical sample impacts the apparent electrode performance. We proposed dark-field microscopy and single molecule fluorescence imaging as tools capable of detangling these effects. Moreover, we established Cresyl Violet as a reporter of single molecule electrochemistry and developed a two-working electrode optical system capable of visualizing single molecule activity. Lastly, we explored the relationships between Au NP size, Cresyl Violet activity and Au NP electrodissolution and found no clear trend between them suggesting the need for more studies to deconvolute these effects and provide meaningful insight into the structure-property relationships. Overall, this dissertation highlights the complexity of single nanoparticle studies and how heterogeneity can be induced from all the components of an electrochemical cell. / Chemistry

Optofluidic flow-through biosensor devices have been in development for fast bio-target detection. Utilizing the fabrication processes developed by the microelectronics industry, these biosensors can be fabricated into lab-on-a-chip devices with a degree of platform portability. This biosensor technology can be used to detect a variety of targets, and is particularly useful for the detection single molecules and nucleic acid strands. Microfabrication also offers the possibility of production at scale, and this will offer a fast detection method for a range of applications with promising economic viability. The development of this technology has advanced to now warrant a descriptive model that will aid in the design of future iterations. The biosensor consists of multiple integrated waveguides and a microfluidic channel. This platform therefore incorporates multiple fields of study: fluorescence, optical waveguiding, microfluidics, and signal counting. This dissertation presents a model theory that integrates all these factors and predicts a biosensor design's sensitivity. The model is validated by comparing simulated tests with physical tests done with fabricated devices. Additionally, the model is used to investigate and comment on designs that have not yet been allocated time and resources to fabricate. Tangentially, an improvement to the fabrication process is investigated and implemented.

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