Waveguide Evanescent-Field Microscopy for Label-Free Monitoring of Biological Nanoparticles: Fabrication, Characterization and Application

Abstract: The recent development of microscopy methods, biological assays and bioanalytical sensors has significantly advanced the understanding of biological systems. Surface-based bioanalytical sensors have in recent years gained increased interest thanks to improvements in sensitivity and simplicity to use. However, most of them, such as quartz crystal microbalance (QCM) and surface plasmon resonance (SPR), provide information based on ensemble averaging of biomolecular interactions. In contrast, with surface-sensitive microscopy methods, biological processes can be resolved down to the level of individual molecular interactions. Total internal reflection fluorescent microscopy is one commonly used surface-sensitive method, reaching sensitivities down to the level of single molecules, but it requires fluorescent labeling of at least one of the interaction partners and is often also hampered by photo bleaching processes. In this thesis, we introduce a new wide-field surface-sensitive microscopy platform, based on a nanofabricated planar optical waveguide design that is capable of label-free evanescent-field microscopy of biological nanoparticles well below 100 nm in diameter. The waveguide generates an evanescent-field at the interface between the core of the waveguide and an aqueous solution, providing a thin sheet of illumination that offers imaging with low background disturbance. The device is presented in two designs, being compatible with either upright or inverted microscopes. The work presented demonstrates how simultaneous monitoring of fluorescence and scattering signals can offer new information about the relation between scattering intensity, refractive index and lipid content of biological nanoparticles, such as exosomes. Further, the microfluidic design allowed not only for convenient liquid handling with dead volumes of a few microliter, it is also showed to aid label-free investigations of the interaction between proteins and individual lipid vesicles, with the latter serving as cell-membrane mimic. With the device also being compatible with formation of fluid supported lipid bilayers, preliminary results suggest that the design will open up a possibility to simultaneously determine the size, scattering intensity and fluorescence emission at the level of individual biological nanoparticles. With this realized, we foresee a broad applicability of the microscopy platform as multidimensional characterization tool for biological nanoparticles and beyond.