| Popis: |
The ability to conduct sub-diffraction optical imaging with super-resolution microscopy has revolutionized imaging in biological research. In this thesis I have utilized super-resolution imaging methods of single molecule localization microscopy (SMLM) and stimulated emission depletion microscopy (STED) to address pertinent questions in various topics of biological and medical research. One of the project deals with imaging motoneuron cells to map the sub-cellular distribution of fused in sarcoma (FUS), a protein involved in the neurodegenerative disease amyotrophic later sclerosis (ALS). By applying cluster analysis on SMLM images, obtained using the direct stochastic optical reconstruction microscopy (dSTORM) method, an increased synaptic accumulation of mutated FUS protein was seen in ALS patient derived motoneurons. Further, sequestering of synaptic proteins Bassoon and Homer1 was also observed in these patient synapses. In the second part of this project, maturation-based variation of FUS localization from post-synapse to pre-synapse was analysed using dSTORM and three-colour DNA-PAINT imaging. In another project the use of dSTORM and STED methods revealed that the antimicrobial peptide LL-37 shows a direct cytotoxic effect on Mycobacterium tuberculosis cells, triggered by cell-wall disruption. Further, it was discovered that LL-37 is internalized by human macrophage cells and localizes in the early endosome vesicles. Finally, using the STED methodology, proof-of-principle super-resolution images of optically cleared post-mortem human brain tissue sections were acquired. |
