Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM).

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

Live-cell imaging under centrifugation characterized the cellular force for nuclear centration in the Caenorhabditis elegans embryo.

Organelles in cells are appropriately positioned, despite crowding in the cytoplasm. However, our understanding of the force required to move large organelles, such as the nucleus, inside the cytoplasm is limited, in part owing to a lack of accurate methods for measurement. We devised a novel method to apply forces to the nucleus of living, wild-type Caenorhabditis elegans embryos to measure the force generated inside the cell. We utilized a centrifuge polarizing microscope (CPM) to apply centrifugal force and orientation-independent differential interference contrast (OI-DIC) microscopy to characterize the mass density of the nucleus and cytoplasm. The cellular forces moving the nucleus toward the cell center increased linearly at ~14 pN/µm depending on the distance from the center. The frictional coefficient was ~1,100 pN s/µm. The measured values were smaller than previously reported estimates for sea urchin embryos. The forces were consistent with the centrosome-organelle mutual pulling model for nuclear centration. Frictional coefficient was reduced when microtubules were shorter or detached from nuclei in mutant embryos, demonstrating the contribution of astral microtubules. Finally, the frictional coefficient was higher than a theoretical estimate, indicating the contribution of uncharacterized properties of the cytoplasm.

Sliding of antiparallel microtubules drives bipolarization of monoastral spindles.

Time-lapse imaging with liquid crystal polarized light (LC-PolScope) and fluorescent speckle microscopy (FSM) enabled this study of spindle microtubules in monoastral spindles that were produced in crane-fly spermatocytes through flattening-induced centrosome displacement. Monoastral spindles are found in several other contexts: after laser ablation of one of a cell's two centrosomes (in the work of Khodjakov et al.), in Drosophila "urchin" mutants (in the works of Heck et al. and of Wilson et al.), in Sciara males (in the works of Fuge and of Metz), and in RNAi variants of Drosophila S2 cells (in the work of Goshima et al.). In all cases, just one pole has a centrosome (the astral pole); the other lacks a centrosome (the anastral pole). Thus, the question: How is the anastral half-spindle, lacking a centrosome, constructed? We learned that monoastral spindles are assembled in two phases: Phase I assembles the astral half-spindle composed of centrosomal microtubules, and Phase II assembles microtubules of the anastral half through extension of new microtubule polymerization outward from the spindle's equatorial mid-zone. That process uses plus ends of existing centrosomal microtubules as guiding templates to assemble anastral microtubules of opposite polarity. Anastral microtubules slide outward with their minus ends leading, thereby establishing proper bipolarity just like in normal biastral spindles that have two centrosomes.

Cellulose assembles into helical bundles of uniform handedness in cell walls with abnormal pectin composition.

Plant cells and organs grow into a remarkable diversity of shapes, as directed by cell walls composed primarily of polysaccharides such as cellulose and multiple structurally distinct pectins. The properties of the cell wall that allow for precise control of morphogenesis are distinct from those of the individual polysaccharide components. For example, cellulose, the primary determinant of cell morphology, is a chiral macromolecule that can self-assemble in vitro into larger-scale structures of consistent chirality, and yet most plant cells do not display consistent chirality in their growth. One interesting exception is the Arabidopsis thaliana rhm1 mutant, which has decreased levels of the pectin rhamnogalacturonan-I and causes conical petal epidermal cells to grow with a left-handed helical twist. Here, we show that in rhm1 the cellulose is bundled into large macrofibrils, unlike the evenly distributed microfibrils of the wild type. This cellulose bundling becomes increasingly severe over time, consistent with cellulose being synthesized normally and then self-associating into macrofibrils. We also show that in the wild type, cellulose is oriented transversely, whereas in rhm1 mutants, the cellulose forms right-handed helices that can account for the helical morphology of the petal cells. Our results indicate that when the composition of pectin is altered, cellulose can form cellular-scale chiral structures in vivo, analogous to the helicoids formed in vitro by cellulose nano-crystals. We propose that an important emergent property of the interplay between rhamnogalacturonan-I and cellulose is to permit the assembly of nonbundled cellulose structures, providing plants flexibility to orient cellulose and direct morphogenesis.

Point spread function of the polarized light field microscope.

We examined the point spread function of the polarized light field microscope and established a computational framework to solve the forward problem in polarized light field imaging, for the purpose of furthering its use as a quantitative tool for measuring three-dimensional maps of the birefringence of transparent objects. We recorded experimental polarized light field images of small calcite crystals and of larger birefringent objects and compared our experimental results to numerical simulations based on polarized light ray tracing. We find good agreement between all our experiments and simulations, which leads us to propose polarized light ray tracing as one solution to the forward problem for the complex, nonlinear imaging mode of the polarized light field microscope. Solutions to the ill-posed inverse problem might be found in analytical methods and/or deep learning approaches that are based on training data generated by the forward solution presented here.

An experimental method to characterize the relationship between aperture image and ray directions in microscope optics.

We propose a direct experimental method to calibrate the relationship between ray directions in object space and their positions in the aperture plane of a light field microscope. The calibration improves the interpretation of light field images, which contain information from both types of image planes, the field plane and the aperture plane of the ray path in the microscope. Our method is based on the diffraction of line gratings of known periodicities and provides accurate results with subpixel resolution. The method can be custom-tailored to most any optical configuration, including standard light microscopy setups, whenever correct mapping between ray parameters in the object/image plane and the aperture plane is needed.

Spatio-angular fluorescence microscopy III. Constrained angular diffusion, polarized excitation, and high-NA imaging.

We investigate rotational diffusion of fluorescent molecules in angular potential wells, the excitation and subsequent emissions from these diffusing molecules, and the imaging of these emissions with high-NA aplanatic optical microscopes. Although dipole emissions only transmit six low-frequency angular components, we show that angular structured illumination can alias higher-frequency angular components into the passband of the imaging system. We show that the number of measurable angular components is limited by the relationships between three time scales: the rotational diffusion time, the fluorescence decay time, and the acquisition time. We demonstrate our model by simulating a numerical phantom in the limits of fast angular diffusion, slow angular diffusion, and weak potentials.

Birefringence Changes of Dendrites in Mouse Hippocampal Slices Revealed with Polarizing Microscopy.

Intrinsic optical signal (IOS) imaging has been widely used to map the patterns of brain activity in vivo in a label-free manner. Traditional IOS refers to changes in light transmission, absorption, reflectance, and scattering of the brain tissue. Here, we use polarized light for IOS imaging to monitor structural changes of cellular and subcellular architectures due to their neuronal activity in isolated brain slices. To reveal fast spatiotemporal changes of subcellular structures associated with neuronal activity, we developed the instantaneous polarized light microscope (PolScope), which allows us to observe birefringence changes in neuronal cells and tissues while stimulating neuronal activity. The instantaneous PolScope records changes in transmission, birefringence, and slow axis orientation in tissue at a high spatial and temporal resolution using a single camera exposure. These capabilities enabled us to correlate polarization-sensitive IOS with traditional IOS on the same preparations. We detected reproducible spatiotemporal changes in both IOSs at the stratum radiatum in mouse hippocampal slices evoked by electrical stimulation at Schaffer collaterals. Upon stimulation, changes in traditional IOS signals were broadly uniform across the area, whereas birefringence imaging revealed local variations not seen in traditional IOS. Locations with high resting birefringence produced larger stimulation-evoked birefringence changes than those produced at low resting birefringence. Local application of glutamate to the synaptic region in CA1 induced an increase in both transmittance and birefringence signals. Blocking synaptic transmission with inhibitors CNQX (for AMPA-type glutamate receptor) and D-APV (for NMDA-type glutamate receptor) reduced the peak amplitude of the optical signals. Changes in both IOSs were enhanced by an inhibitor of the membranous glutamate transporter, DL-TBOA. Our results indicate that the detection of activity-induced structural changes of the subcellular architecture in dendrites is possible in a label-free manner.

Spatio-angular fluorescence microscopy I. Basic theory.

We introduce the basic elements of a spatio-angular theory of fluorescence microscopy, providing a unified framework for analyzing systems that image single fluorescent dipoles and ensembles of overlapping dipoles that label biological molecules. We model an aplanatic microscope imaging an ensemble of fluorescent dipoles as a linear Hilbert-space operator, and we show that the operator takes a particularly convenient form when expressed in a basis of complex exponentials and spherical harmonics-a form we call the dipole spatio-angular transfer function. We discuss the implications of our analysis for all quantitative fluorescence microscopy studies and lay out a path toward a complete theory.

Spatio-angular fluorescence microscopy II. Paraxial 4f imaging.

We investigate the properties of a single-view fluorescence microscope in a 4f geometry when imaging fluorescent dipoles without using the monopole or scalar approximations. We show that this imaging system has a spatio-angular band limit, and we exploit the band limit to perform efficient simulations. Notably, we show that information about the out-of-plane orientation of ensembles of in-focus fluorophores is recorded by paraxial fluorescence microscopes. Additionally, we show that the monopole approximation may cause biased estimates of fluorophore concentrations, but these biases are small when the sample contains either many randomly oriented fluorophores in each resolvable volume or unconstrained rotating fluorophores.

Bacillus subtilis cell diameter is determined by the opposing actions of two distinct cell wall synthetic systems.

Rod-shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: the Rod complex moves around the cell circumference, whereas class A penicillin-binding proteins (aPBPs) do not. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each affects the growth and width of Bacillus subtilis as well as the mechanical anisotropy and orientation of material within their sacculi. Rod width is not determined by MreB, rather it depends on the balance between the systems: the Rod complex reduces diameter, whereas aPBPs increase it. Increased Rod-complex activity correlates with an increased density of directional MreB filaments and a greater fraction of directional PBP2a enzymes. This increased circumferential synthesis increases the relative quantity of oriented material within the sacculi, making them more resistant to stretching across their width, thereby reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems builds and maintains rods of different widths. Escherichia coli Rod mutants also show the same correlation between width and directional MreB filament density, suggesting this model may be generalizable to bacteria that elongate via the Rod complex.

Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites.

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.

Single-fluorophore orientation determination with multiview polarized illumination: modeling and microscope design.

We investigate the use of polarized illumination in multiview microscopes for determining the orientation of single-molecule fluorescence transition dipoles. First, we relate the orientation of single dipoles to measurable intensities in multiview microscopes and develop an information-theoretic metric-the solid-angle uncertainty-to compare the ability of multiview microscopes to estimate the orientation of single dipoles. Next, we compare a broad class of microscopes using this metric-single- and dual-view microscopes with varying illumination polarization, illumination numerical aperture (NA), detection NA, obliquity, asymmetry, and exposure. We find that multi-view microscopes can measure all dipole orientations, while the orientations measurable with single-view microscopes is halved because of symmetries in the detection process. We also find that choosing a small illumination NA and a large detection NA are good design choices, that multiview microscopes can benefit from oblique illumination and detection, and that asymmetric NA microscopes can benefit from exposure asymmetry.

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration.

Integrin αß heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding ß-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, µm-scale measurements.

Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments.

The actomyosin ring generates force to ingress the cytokinetic cleavage furrow in animal cells, yet its filament organization and the mechanism of contractility is not well understood. We quantified actin filament order in human cells using fluorescence polarization microscopy and found that cleavage furrow ingression initiates by contraction of an equatorial actin network with randomly oriented filaments. The network subsequently gradually reoriented actin filaments along the cell equator. This strictly depended on myosin II activity, suggesting local network reorganization by mechanical forces. Cortical laser microsurgery revealed that during cytokinesis progression, mechanical tension increased substantially along the direction of the cell equator, while the network contracted laterally along the pole-to-pole axis without a detectable increase in tension. Our data suggest that an asymmetric increase in cortical tension promotes filament reorientation along the cytokinetic cleavage furrow, which might have implications for diverse other biological processes involving actomyosin rings.

Quantification of collagen organization in histopathology samples using liquid crystal based polarization microscopy.

A number of histopathology studies have utilized the label free microscopy method of Second Harmonic Generation (SHG) to investigate collagen organization in disease onset and progression. Here we explored an alternative label free imaging approach, LC-PolScope that is based on liquid crystal based polarized light imaging. We demonstrated that this more accessible technology has the ability to visualize all fibers of interest and has a good to excellent correlation between SHG and LC-PolScope measurements in fibrillar collagen orientation and alignment. This study supports that LC-PolScope is a viable alternative to SHG for label free collagen organization measurements in thin histology sections.

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions.

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVß3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.

Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy.

Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis.

Achiral symmetry breaking and positive Gaussian modulus lead to scalloped colloidal membranes.

In the presence of a nonadsorbing polymer, monodisperse rod-like particles assemble into colloidal membranes, which are one-rod-length-thick liquid-like monolayers of aligned rods. Unlike 3D edgeless bilayer vesicles, colloidal monolayer membranes form open structures with an exposed edge, thus presenting an opportunity to study elasticity of fluid sheets. Membranes assembled from single-component chiral rods form flat disks with uniform edge twist. In comparison, membranes composed of a mixture of rods with opposite chiralities can have the edge twist of either handedness. In this limit, disk-shaped membranes become unstable, instead forming structures with scalloped edges, where two adjacent lobes with opposite handedness are separated by a cusp-shaped point defect. Such membranes adopt a 3D configuration, with cusp defects alternatively located above and below the membrane plane. In the achiral regime, the cusp defects have repulsive interactions, but away from this limit we measure effective long-ranged attractive binding. A phenomenological model shows that the increase in the edge energy of scalloped membranes is compensated by concomitant decrease in the deformation energy due to Gaussian curvature associated with scalloped edges, demonstrating that colloidal membranes have positive Gaussian modulus. A simple excluded volume argument predicts the sign and magnitude of the Gaussian curvature modulus that is in agreement with experimental measurements. Our results provide insight into how the interplay between membrane elasticity, geometrical frustration, and achiral symmetry breaking can be used to fold colloidal membranes into 3D shapes.