3 research outputs found
Metal Ion Based Probes for Imaging
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+
Visualization of Cellular Components in a Mammalian Cell with Liquid-Cell Transmission Electron Microscopy
We present liquid-cell transmission electron microscopy (liquid-cell TEM) imaging of fixed and non-fixed prostate cancer cells (PC3 and LNCaP) with high resolution in a custom developed silicon nitride liquid cell. Fixed PC3 cells were imaged for 90–120 min without any discernable damage. High contrast on the cellular structures was obtained even at low electron doses (~2.5 e-/nm2 per image). The images show distinct structures of cell compartments (nuclei and nucleoli) and cell boundaries without any further sample embedding, dehydration, or staining. Furthermore, we observed dynamics of vesicles trafficking from the cell membrane in consecutive still frames in a non-fixed cell. Our findings show that liquid-cell TEM, operated at low electron dose, is an excellent tool to investigate dynamic events in non-fixed cells with enough spatial resolution (few nm) and natural amplitude contrast to follow key intracellular processes
Self-assembling peptides imaged by correlated liquid cell transmission electron microscopy and MALDI-imaging mass spectrometry
We describe the observation of stimuli-induced peptide-based nanoscale assemblies by liquid cell transmission electron microscopy (LCTEM). LCTEM offers the opportunity to directly image nanoscale materials in liquid. Despite broad interest in characterizing biological phenomena, electron beam-induced damage remains a significant problem. Concurrently, methods for verifying chemical structure during or following an LCTEM experiment have been few, with key examples limited to electron diffraction or elemental analysis of crystalline materials; this strategy is not translatable to biopolymers observed in nature. In this proof-of-concept study, oligomeric peptides are biologically or chemically stimulated within the liquid cell in a TEM to assemble into nanostructures. The resulting materials are analyzed by MALDI-imaging mass spectrometry (MALDI-IMS) to verify their identity. This approach confirms whether higher-order assemblies observed by LCTEM consist of intact peptides, verifying that observations made during the in situ experiment are because of those same peptides and not aberrant electron beam damage effects