Single-molecule imaging of the biophysics of molecular interactions with precision and control, in cell-like conditions, and without tethers

Abstract Molecular biology is messy and complex. The future of life sciences research, drug development, and many fields depends on our ability to unravel the complex, biophysical phenomena that underlie cellular function with a finer level of resolution. Using current technologies, it is challenging to conduct quantitative measurements that can reveal the complexity of life at the molecular scale. This article reviews the invention, development, and research applications of Convex Lens-induced Confinement (CLiC) microscopy, which is a method to image molecular interactions one molecule at a time, while emulating “cell like” conditions, with precision and control. By mechanically confining molecules to the field of view, CLiC can eliminate the complexity and potential biases inherent to “tethering” molecules. Looking forwards, CLiC is being applied to emerging areas of exploration where single-molecule resolution can be transformative, including visualizing nanoparticle and protein dynamics, CRISPR-Cas9 targeting dynamics, and therapeutics applications.