Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30°of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.single-molecule orientation | live cell imaging | polarized fluorescence | actin | septin T he generation and dissolution of order within populations of biological molecules are central to almost all cellular processes. The emergence of ordered arrays of biological molecules is manifested in lipid membranes, DNA, the cytoskeleton, and many other molecular assemblies in the cytoplasm of the living cell. Polarization-resolved fluorescence imaging of densely labeled assemblies has successfully probed their net order and architectural dynamics (1-5). In addition, tracking of sparsely labeled cytoskeletal networks (6-9) has illuminated how turnover of individual molecules enables transitions in the spatial organization of cytoskeletal networks. We have combined tracking of both orientation and position of single fluorophores associated with cystoskeletal proteins in live cells to analyze the molecular order within cytoskeletal assemblies.Fluorophores, including the GFP, emit fluorescence by radiating light as dipoles. The light emitted from a single dipole is fully polarized, with most of the energy polarized along the dipole axis. For example, the absorption and emission dipole axes of GFP chromophores are fixed within the GFP molecule as was found in GFP crystals (10, 11). Therefore, fluorescent labels can report the orientation of biomolecules as long as the labels are rigidly bound to the biomolecules, ...

We have developed an imaging system for 3D time-lapse polarization microscopy of living biological samples. Polarization imaging reveals the position, alignment and orientation of submicroscopic features in label-free as well as fluorescently labeled specimens. Optical anisotropies are calculated from a series of images where the sample is illuminated by light of different polarization states. Due to the number of images necessary to collect both multiple polarization states and multiple focal planes, 3D polarization imaging is most often prohibitively slow. Our MF-PolScope system employs multifocus optics to form an instantaneous 3D image of up to 25 simultaneous focal-planes. We describe this optical system and show examples of 3D multi-focus polarization imaging of biological samples, including a protein assembly study in budding yeast cells.

Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of polarized fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30°of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.single-molecule orientation | live cell imaging | polarized fluorescence | actin | septin T he generation and dissolution of order within populations of biological molecules are central to almost all cellular processes. The emergence of ordered arrays of biological molecules is manifested in lipid membranes, DNA, the cytoskeleton, and many other molecular assemblies in the cytoplasm of the living cell. Polarization-resolved fluorescence imaging of densely labeled assemblies has successfully probed their net order and architectural dynamics (1-5). In addition, tracking of sparsely labeled cytoskeletal networks (6-9) has illuminated how turnover of individual molecules enables transitions in the spatial organization of cytoskeletal networks. We have combined tracking of both orientation and position of single fluorophores associated with cystoskeletal proteins in live cells to analyze the molecular order within cytoskeletal assemblies.Fluorophores, including the GFP, emit fluorescence by radiating light as dipoles. The light emitted from a single dipole is fully polarized, with most of the energy polarized along the dipole axis. For example, the absorption and emission dipole axes of GFP chromophores are fixed within the GFP molecule as was found in GFP crystals (10, 11). Therefore, fluorescent labels can report the orientation of biomolecules as long as the labels are rigidly bound to the biomolecules, ...