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A fundamental challenge with fluorophore orientation measurement is degeneracy, which is the inability to distinguish between multiple unique fluorophore orientations. Techniques exist for the non-degenerate measurement of the orientations of single, static fluorophores. However, such techniques are unsuitable for densely labeled and/or dynamic samples common to biological research. Accordingly, a rapid, widefield microscopy technique that can measure orientation parameters for ensembles of fluorophores in a non-degenerate manner is desirable. We propose that exciting samples with polarized light and multiple incidence angles could enable such a technique. We use Monte Carlo simulations to validate this approach for specific axially symmetric ensembles of fluorophores and obtain optimal experimental parameters for its future implementation.

Podosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces.

Inspired by biological motor proteins, that efficiently convert chemical fuel to unidirectional motion, there has been considerable interest in developing synthetic analogues. Among the synthetic motors created thus far, DNA motors that undertake discrete steps on RNA tracks have shown the greatest promise. Nonetheless, DNA nanomotors lack intrinsic directionality, are low speed and take a limited number of steps prior to stalling or dissociation. Herein, we report the first example of a highly tunable DNA origami motor that moves linearly over micron distances at an average speed of 40 nm/min. Importantly, nanomotors move unidirectionally without intervention through an external force field or a patterned track. Because DNA origami enables precise testing of nanoscale structure‐function relationships, we were able to experimentally study the role of motor shape, chassis flexibility, leg distribution, and total number of legs in tuning performance. An anisotropic rigid chassis coupled with a high density of legs maximizes nanomotor speed and endurance.

Inspired by biological motor proteins, that efficiently convert chemical fuel to unidirectional motion, there has been considerable interest in developing synthetic analogues. Among the synthetic motors created thus far, DNA motors that undertake discrete steps on RNA tracks have shown the greatest promise. Nonetheless, DNA nanomotors lack intrinsic directionality, are low speed and take a limited number of steps prior to stalling or dissociation. Herein, we report the first example of a highly tunable DNA origami motor that moves linearly over micron distances at an average speed of 40 nm/min. Importantly, nanomotors move unidirectionally without intervention through an external force field or a patterned track. Because DNA origami enables precise testing of nanoscale structure‐function relationships, we were able to experimentally study the role of motor shape, chassis flexibility, leg distribution, and total number of legs in tuning performance. An anisotropic rigid chassis coupled with a high density of legs maximizes nanomotor speed and endurance.