In Honor of W.E. Moerner: Confining Molecules for Single‐Molecule Spectroscopy

Abstract

Single‐molecule spectroscopy provides a wealth of information on the dynamics and interactions of complex biological molecules. Yet these measurements are extremely challenging, partly because Brownian motion prevents molecules in free solution from remaining stationary. Here we describe several techniques that our lab has developed for confining single molecules for optical spectroscopy. These alternatives to surface immobilization provide confinement that is gentle enough to minimize perturbations to the molecule, but strong enough to allow long‐time imaging of single fluorescent molecules, often in the presence of a high fluorescent background.

Mass Action at the Single-Molecule Level

“…The device enables highly parallel, long-term, and attachment-free studies of single molecules or small numbers of molecules confined in nanofabricated chambers. Confinement has previously been used in single-molecule studies, but under conditions where the confined region was large compared to the optical wavelength or where the confinement was in self-assembled structures lacking precisely defined sizes and locations. − Here we achieved sufficiently tight confinement that (a) the radius of confinement was small compared to the mean separation of reactant molecules in bulk solution and (b) the radius of confinement was small enough that all possible pairwise intermolecular collisions occurred on an experimentally accessible time scale. This qualitatively new regime led to several apparent deviations from the law of mass action, which are reconciled by a statistical description of reactivity in confinement.…”

“…Dimple Machine technology provides a possible resolution to many challenges associated with single-molecule measurements in solution . Molecules in free solution are typically observed for fleeting moments due to diffusion, while surface tethering may disrupt molecular function.…”

Precision platform for convex lens-induced confinement microscopy

“…15 However, surfaceimmobilization or applied flow can constrain the observed range of molecular conformations and affect molecular activity. 3,17 For example, the superhelicity-driven dynamics of physiological DNA, which govern local unwinding of promoter sites to which transcription factors bind, 18 would not be reflected in typical molecular search experiments performed using linear DNA.…”

The Cat That Caught the Canary: What To Do with Single-Molecule Trapping

“…The ABEL trap is just one of several technologies under development to facilitate studies on single molecules in free solution . Significant information can be obtained by confining molecules between parallel walls, or in thin capillaries, lipid vesicles, nanofabricated zero-mode waveguides, or water-in-oil hydrosomes .…”

“…The ABEL trap is just one of several technologies under development to facilitate studies on single molecules in free solution. 18 Significant information can be obtained by confining molecules between parallel walls, 19 or in thin capillaries, 20 lipid vesicles, 21 nanofabricated zero-mode waveguides, 22 or water-in-oil hydrosomes. 23 These devices achieve confinement through purely mechanical means, with the attendant decrease in complexity relative to the ABEL trap, but also a loss of high-resolution electrokinetic data.…”