Fabrication, development and characterization of a waveguide microscopy device for biological applications

Abstract: Our understanding of biological systems has advanced significantly thanks to the development of microscopy methods and suitable biological assays. Surface based methods have increasingly gained interest thanks to their superior sensitivity and ease of use. Still, 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, surface-sensitive microscopy methods provide the possibility to study biological processes on an individual biomolecular basis. Total internal reflection fluorescent (TIRF) microscopy is a surface-sensitive microscopy method that reaches sensitivities down to the level of single molecules, but it is limited by the need to fluorescently label the interaction partners. On the contrary, waveguide-based evanescent-light scattering microscopy provides a label-free surface-sensitive imaging technique that can also benefit from fluorescent labeling if desired. Here we present ongoing efforts to further develop a waveguide-based platform for evanescent light-scattering microscopy. By adopting the fabrication and processing steps to a transparent substrate and adding compatible microfluidics, we can now use high NA oil-immersion objectives thereby collecting more light and resolve details that could not be resolved when fabricated on an opaque substrate. Further, well-controlled microfluidic handling makes it possible to perform more complicated experiments and extract data on interaction dynamics. Moreover an image processing code has been developed that can be applied to the different experimental sequences, including both waveguide fluorescent and scattering modes, and thereby extract otherwise hidden information in the experimental data.