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要旨:
In the framework of this thesis, metal ions were used for two different approaches for the application as bimodal probes for light and electron microscopy.
Gold nanoparticles (AuNPs) were used in the first strategy to establish in-liquid TEM. The AuNPs were used to image DNA-hybridization. Moreover, mammalian cells were imaged using in-liquid TEM. Subcellular structures such as nucleus, nucleoli, and cell membranes were visible without additional staining or sample preparation. It was also possible to perform live cell imaging of prostate cancer cells (PC3 and LNCaP) using in-liquid TEM. Dynamics of subcellular structures could be observed. Ribonucleotide-functionalized AuNPs were also used for live cell imaging.
The second approach used Sm3+ ions and the enzyme aequorin (AQ). AQ was engineered towards a bimodal probe for light and electron microscopy. The enzyme emits light upon metal ion binding. The natural metal ion inducing luminescence is Ca2+. The engineering yielded a new variant TS A123W S125E that had 40-fold improved affinity for Sm3+ compared to the parental thermostabilized AQ TS. The Ca2+ affinity decreased 30-fold.
Binding of Sm3+ of the new variant TS A123W S125E could induce contrast in conventional TEM analysis. Additionally it showed luminescence in cell tests with PC3 cells for both Sm3+ and Ca2+.
