Single Fluorogens and Orientation-Localization Microscopy for Quantifying Chemical and Biomolecular Dynamics at the Nanoscale.

Many chemical systems look uniform only because ensemble measurements average over their most interesting molecules. Electron-transfer rates vary across electrode surfaces; lipid membranes contain nanodomains with distinct packing and fluidity; peptide aggregates exhibit local polymorphism; and biomolecular condensates contain transient networks of interactions that are blurred in ensemble images. A central challenge in chemical imaging is not simply to see smaller structures, but to measure chemical variables such as polarity, redox potential, and molecular confinement at the single-molecule level. In this Account, we describe how fluorogens, molecules whose brightness, blinking, spectral shifts, orientation, rotational mobility, and motion are directly shaped by their surroundings, can be integrated with single-molecule orientation-localization microscopy (SMOLM) to produce multidimensional images of nanoscale chemistry. The key idea is to treat a fluorophore as a molecular sensor instead of as a passive label. Its brightness and blinking can report local polarity or redox state; its position identifies where the event occurs; its orientation and rotational freedom reveal local order and confinement; and its trajectory reports transport or local viscoelasticity. By constructing single-molecule (SM) polarization-sensitive microscopes, we measure molecular orientation and wobble alongside nanoscale position and intensity. Thus, SMOLM provides the optical and computational machinery to extract multidimensional chemical observables from single fluorogens. We first highlight SM electrochemical imaging, in which redox-sensitive fluorophores report local midpoint potentials, revealing spatial variations in electron-transfer behavior hidden by ensemble averaging and showing how redox mediators can homogenize and shift fluorophore reduction. We then turn to lipid membranes, where solvatochromic lipophilic probes such as Nile red and merocyanine 540 sense local packing, polarity, and fluidity. By measuring both rotational and translational dynamics, SMOLM shows that cholesterol-rich membrane regions can constrain molecular orientation while permitting heterogeneous, jump-like lateral motion and that cellular membrane curvature and composition generate spatially varying orientational order. In peptide and amyloid assemblies, transiently binding fluorogens convert surface chemistry into nanoscale structural information. Nile red orientations report the alignment, disorder, and polymorphism of fibrillar architectures, distinguishing mature regions from newly deposited or disordered branches and revealing helical bilayer-like organization that is inaccessible to position-only super-resolution microscopy. Finally, we discuss biomolecular condensates, where environmentally sensitive fluorogens expose hidden nanoscale organization within droplets that often appear homogeneous by diffraction-limited imaging. SM trajectories identify hydrophobic hubs, speed-resolved maps reveal confined microphases, fluorogen mobility senses sequence variations in intrinsically disordered proteins, and SM tracking uncovers anisotropic diffusion along RNA condensate boundaries. Together, these examples show that the richest information in fluorescence microscopy often lies not only in where a molecule is but also in how it behaves. The broader message is that fluorophores should be chosen not only for brightness or photostability but also for the chemical variables they can report. By uniting fluorogen chemistry, optical design, and computational imaging, single-fluorogen orientation-localization microscopy provides a route to image chemical environments, molecular organization, and nonequilibrium dynamics with nanoscale precision. Essentially, single fluorogens emerge as quantitative reporters of chemistry in action.