Single-molecule spectroscopy & super-resolution microscopy at the biochemistry bench

Moya Muñoz, Gustavo Gabriel (2025): Single-molecule spectroscopy & super-resolution microscopy at the biochemistry bench. Dissertation, LMU München: Fakultät für Biologie

Abstract

Single-molecule spectroscopy and super-resolution microscopy offer valuable insights into molecular dynamics but have been limited by high costs and technical complexity. These tools are mostly accessible to specialized labs with custom-built systems. This work aims to make them more affordable and accessible to a wider range of researchers, including those in smaller or resource-limited labs. A major challenge in single-molecule experiments is the variability in experimental setups, often due to the use of home-built systems, a limitation common across all single-molecule techniques. In the context of smFRET, which this study focused on, applying established data correction routines enabled reliable and comparable results across different setups. The most critical parameter influencing data accuracy was the gamma factor, which accounts for differences in the quantum yields of the donor and acceptor fluorophores, as well as the wavelength-dependent detection efficiencies of the point detectors. However, its overall impact was minimal given the typical FRET efficiency differences observed in biomolecules, underscoring the importance of thoughtful protein and fluorophore design to minimize variability. Comparisons with other techniques, like Pulsed Electron-Electron Double Resonance (PELDOR) and anomalous X-ray scattering interferometry (AXSI), confirmed that smFRET provides consistent distance measurements. Discrepancies arose due to fluorophore-protein interactions but could be mitigated through careful experimental design. A key development of this work is Brick-MIC, an affordable, open-source platform for single-molecule experiments. Built with 3D printing and open-source software, Brick-MIC allows researchers to customize setups at a fraction of traditional costs. It supports techniques like smFRET, fluorescence correlation spectroscopy (FCS), and super-resolution imaging, making these tools more accessible to the scientific community. In a simplified iteration, a blue-green FRET system was created using a 488 nm laser, making it cost-effective while still providing valuable insights into biomolecular conformational changes. This system, while lacking stoichiometric information, enables the observation of biomolecule movements, catering to application-driven studies. Additionally, Brick-MIC was applied to nanoparticle detection, specifically identifying SARS-CoV-2 virus particles. By combining microfluidics, fluorescence correlation spectroscopy, and dual-layer detection strategies, this work enabled rapid and specific virus detection, demonstrating the practical applications of this affordable platform in diagnostics and public health.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: smFRET, FCS, SMLM, 3D-Printing, microscopy
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 5. Juni 2025
• 1. Bericht­erstatter:in: Cordes, Thorben