Correlative Chemical Imaging of Nanoscale Subcellular Structures

Sammanfattning: Chemical imaging can elucidate complex mechanisms, relationships, and components of biological samples. For example, it can reveal properties such as chemical composition, chemical structure, reactivity, and topography. Several imaging techniques exist, each providing different types of information. Yet, no single technique can comprehensively characterize a sample. Having a holistic profile often requires correlating complementary methods; for example, scanning electron microscopy (SEM) can be combined with secondary ion mass spectrometry (SIMS) imaging to obtain insights on both the physical topography (via SEM) and the chemical composition (via SIMS) of a sample surface. This approach is referred to as correlative imaging. Correlative chemical imaging is applicable in many scientific fields, such as biology, chemistry, geology, and material science. Among the wide variety of modern imaging techniques that exist, nanoscale SIMS (NanoSIMS) emerges as a powerful tool, having seen growing applications, especially in biochemistry and cell biology. To this end, it can be used for the detection of isotopically labeled material in a sample and provides the chemical composition of the sample surface with high lateral resolution (down to 50 nm), sensitivity (ppm-ppb range), and mass resolution (up to 10000). By using an isotopic label, target molecules in the sample can be studied, although unlabeled samples can be used in some cases. NanoSIMS presents some limitations; for example, it usually cannot discern the ultrastructure of very small, intricate sample details (e.g., subcellular ultrastructure). Therefore, NanoSIMS is often correlated with additional imaging techniques, such as microscopy, to push its capabilities and overcome its shortcomings. In the papers which are part of this thesis, NanoSIMS imaging was correlated with either electron or light microscopy to address different biological questions. To discern nanoscale subcellular ultrastructures, transmission electron microscopy (TEM) was employed, and to localize an organelle labeled with an antibody and a fluorescent tag, STED microscopy was used. In paper I, NanoSIMS was employed to detect 13C-dopamine in PC12 cells, and the images correlated with TEM to localize the dopamine within large dense core vesicles (LDCVs). In paper II, NanoSIMS was correlated with stimulated emission-depletion (STED) microscopy to localize endoplasmic reticulum stress-induced stress granules (SGs) in neuronal progenitor cells (NPCs) incubated with an isotopically labeled amino acid, and to characterize their protein turnover by changes in isotopic enrichment. In paper III, I investigated the role of vesicle size in the dynamics of partial release exocytosis events of PC12 cells by correlating TEM and NanoSIMS imaging data. In paper IV, NanoSIMS and TEM were correlated to look at the subcellular protein turnover in NPCs using different isotopically labeled amino acids and time-points. Overall, these studies demonstrate the importance of adequate correlative imaging strategies, and the variety of biological aims that can be achieved through different correlative chemical imaging approaches.