Kern, KlausEdte, Moritz Alexander2025-09-102025-09-102025-09-10202510.5075/epfl-thesis-11353https://infoscience.epfl.ch/handle/20.500.14299/253935Understanding the structure of biomolecules at the single-molecule level is crucial for elucidating their biological function. Traditional structural biology techniques such as X-ray crystallography and cryo-electron microscopy rely on ensemble averaging and often struggle to resolve flexible or non-crystalline systems. On the other hand, nuclear magnetic resonance spectroscopy (NMR) provides valuable insights into the structure and dynamics of molecular systems, but its application becomes increasingly challenging as the systems become larger and often requires complementary data to introduce accurate constraints. This highlights the need for a complementary approach that enables direct imaging of individual biomolecules. This thesis presents the development and application of a novel experimental platform that combines native electrospray ion beam deposition (ES IBD) with low-energy electron holography (LEEH) to directly image individual biomolecules in native-like conformations. The integrated system enables mass- and shape-selective landing onto ultraclean substrates, both at room temperature and under cryogenic conditions, followed by sub-nanometer resolution imaging. By advancing both the hardware and data analysis capabilities of the ES IBD-LEEH workflow, this work significantly increases acquisition speed and improves the fidelity of biomolecule imaging. Applications include the successful deposition and imaging of Beta-Galactosidase, DNA, and mucin molecules, highlighting the platforms capability to resolve fine structural details and conformational variability. These results underscore the potential of this method to complement traditional structural biology techniques, particularly in the study of flexible biomolecules that remain difficult to characterize by conventional means.enLow-energy electron holography (LEEH)Electrospray ion beam deposition (ES IBD)Single-molecule imagingStructural biologyCryogenic depositionDNABeta-GalactosidaseMucinsIon mobilityMass spectrometrythesis::doctoral thesis