All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A’s interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.