Combining Fluorescence and Nanofluidic Scattering Microscopy For Biomolecule Characterization

Abstract

Characterization of single biomolecules is fundamental for understanding biological processes at the molecular level. Although fluorescence microscopy using nanofluidic devices has been a longstanding workhorse for the study of single biomolecules, crucial limitations exist, with the main being alterations in structural conformation due to fluorophore binding. Techniques based on detection of scattering light, such as iSCAT (Interferometric scattering microscopy) microscopy, have emerged and evolved to label-free biolomecular probing techniques. Nanofluidic scattering microscopy (NSM), a technique relying on freely diffusing biomolecules inside nanofluidic structures, has also been developed the last years with impressive results as a reliable label-free characterization method. This thesis investigates the applicability of NSM as a label-free technique for the characterization of single DNA molecules confined inside nanochannels, using fluorescence microscopy as a reference method. Both measurements were performed under comparable experimental conditions to evaluate the capability of both techniques in determining DNA extension. In parallel, a dedicated analysis algorithm was developed for the fluorescence measurement technique, including automated molecular detection, trajectory extraction, and DNA length estimation using errorfunction fitting. Measurements were performed on DNA samples prepared in 0.5×TBE and 0.05×TBE. Fluorescence microscopy showed larger and more variable molecular extensions in 0.05×TBE, while NSM yielded similar median extensions under both conditions, with a broader distribution in 0.05×TBE. Correlated measurements showed a positive relationship between the two methods, although individual discrepancies and potential selection bias require further investigation before quantitative agreement can be established. The fluorescence analysis also requires further optimization for reliable measurements of DNA extension fluctuations. This work establishes an experimental and analytical framework for simultaneous fluorescence microscopy and NSM measurements of confined single DNA molecules.