Convex Lens-Induced Confinement: Enabling New Biophysical Measurements Under Previously Inaccessible Conditions

A wide range of physiological processes rely on weak intermolecular interactions that occur at high concentration, or over long time periods. Probing such interactions presents a challenge to fluorescence microscopy, the work horse for resolving biological processes at the molecular scale. To address this challenge, I present a novel and practical fluorescence imaging technique, convex lens induced confinement (CLIC), which enables new biophysical measurements under previously inaccessible conditions. By confining the molecules within a thin chamber, the CLIC technique dramatically decreases background fluorescence while increasing imaging time. I show that the enhanced background rejection can be used to probe the weak interaction between myosin motor proteins and the requisite micromolar concentration solutions of ATP that fuel their motion. Further, I am using the 10,000 fold increase in per-molecule observation time of freely diffusing DNA to study their interaction with the DNA-binding protein HMGB1 in free solution. The CLIC system also provides direct means to determine molecular size, which I demonstrate for protein and DNA molecules.