Permittivity tensor imaging: modular label-free imaging of 3D dry mass and 3D orientation at high resolution.

The dry mass and the orientation of biomolecules can be imaged without a label by measuring their permittivity tensor (PT), which describes how biomolecules affect the phase and polarization of light. Three-dimensional (3D) imaging of PT has been challenging. We present a label-free computational microscopy technique, PT imaging (PTI), for the 3D measurement of PT. PTI encodes the invisible PT into images using oblique illumination, polarization-sensitive detection and volumetric sampling. PT is decoded from the data with a vectorial imaging model and a multi-channel inverse algorithm, assuming uniaxial symmetry in each voxel. We demonstrate high-resolution imaging of PT of isotropic beads, anisotropic glass targets, mouse brain tissue, infected cells and histology slides. PTI outperforms previous label-free imaging techniques such as vector tomography, ptychography and light-field imaging in resolving the 3D orientation and symmetry of organelles, cells and tissue. We provide open-source software and modular hardware to enable the adoption of the method.

Building momentum through networks: Bioimaging across the Americas.

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates.

Contact guidance is a powerful topographical cue that induces persistent directional cell migration. Healthy tissue stroma is characterized by a meshwork of wavy extracellular matrix (ECM) fiber bundles, whereas metastasis-prone stroma exhibit less wavy, more linear fibers. The latter topography correlates with poor prognosis, whereas more wavy bundles correlate with benign tumors. We designed nanotopographic ECM-coated substrates that mimic collagen fibril waveforms seen in tumors and healthy tissues to determine how these nanotopographies may regulate cancer cell polarization and migration machineries. Cell polarization and directional migration were inhibited by fibril-like wave substrates above a threshold amplitude. Although polarity signals and actin nucleation factors were required for polarization and migration on low-amplitude wave substrates, they did not localize to cell leading edges. Instead, these factors localized to wave peaks, creating multiple "cryptic leading edges" within cells. On high-amplitude wave substrates, retrograde flow from large cryptic leading edges depolarized stress fibers and focal adhesions and inhibited cell migration. On low-amplitude wave substrates, actomyosin contractility overrode the small cryptic leading edges and drove stress fiber and focal adhesion orientation along the wave axis to mediate directional migration. Cancer cells of different intrinsic contractility depolarized at different wave amplitudes, and cell polarization response to wavy substrates could be tuned by manipulating contractility. We propose that ECM fibril waveforms with sufficiently high amplitude around tumors may serve as "cell polarization barriers," decreasing directional migration of tumor cells, which could be overcome by up-regulation of tumor cell contractility.

Visualizing Sphingosine-1-Phosphate Receptor 1(S1P1) Signaling During Central Nervous System De- and Remyelination.

Multiple sclerosis (MS) is an inflammatory-demyelinating disease of the central nervous system (CNS) mediated by aberrant auto-reactive immune responses. The current immune-modulatory therapies are unable to protect and repair immune-mediated neural tissue damage. One of the therapeutic targets in MS is the sphingosine-1-phosphate (S1P) pathway which signals via sphingosine-1-phosphate receptors 1-5 (S1P1-5). S1P receptors are expressed predominantly on immune and CNS cells. Considering the potential neuroprotective properties of S1P signaling, we utilized S1P1-GFP (Green fluorescent protein) reporter mice in the cuprizone-induced demyelination model to investigate in vivo S1P - S1P1 signaling in the CNS. We observed S1P1 signaling in a subset of neural stem cells in the subventricular zone (SVZ) during demyelination. During remyelination, S1P1 signaling is expressed in oligodendrocyte progenitor cells in the SVZ and mature oligodendrocytes in the medial corpus callosum (MCC). In the cuprizone model, we did not observe S1P1 signaling in neurons and astrocytes. We also observed ß-arrestin-dependent S1P1 signaling in lymphocytes during demyelination and CNS inflammation. Our findings reveal ß-arrestin-dependent S1P1 signaling in oligodendrocyte lineage cells implying a role of S1P1 signaling in remyelination.

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.

Dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live 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.

Partially coherent microscope in phase space.

Explicit expressions are presented for different phase-space representations (mutual intensity, Wigner distribution function, and ambiguity function) of the partially coherent image wave field in a microscope system. These are separated into system- and object-dependent parts. The partially coherent image in phase space can be described in terms of different 6D system-dependent kernels, all Fourier transforms of the system mutual spectrum, the region of overlap of two displaced objective pupils and the effective source. The image intensity can be expressed in terms of a 4D kernel, the convolution in spatial frequency of the source, and the Wigner distribution function of the objective pupil, given by a marginal of, or a section through, the respective phase-space kernels.

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.

Septin assemblies form by diffusion-driven annealing on membranes.

Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.

MultiFocus Polarization Microscope (MF-PolScope) for 3D polarization imaging of up to 25 focal planes simultaneously.

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.

Polarized light microscopy in reproductive and developmental biology.

The polarized light microscope reveals orientational order in native molecular structures inside living cells, tissues, and whole organisms. It is a powerful tool used to monitor and analyze the early developmental stages of organisms that lend themselves to microscopic observations. In this article, we briefly discuss the components specific to a traditional polarizing microscope and some historically important observations on: chromosome packing in the sperm head, the first zygote division of the sea urchin, and differentiation initiated by the first asymmetric cell division in the sand dollar. We then introduce the LC-PolScope and describe its use for measuring birefringence and polarized fluorescence in living cells and tissues. Applications range from the enucleation of mouse oocytes to analyzing the polarized fluorescence of the water strider acrosome. We end with new results on the birefringence of the developing chick brain, which we analyzed between developmental stages of days 12-20.

Mantis: high-throughput 4D imaging and analysis of the molecular and physical architecture of cells.

High-throughput dynamic imaging of cells and organelles is essential for understanding complex cellular responses. We report Mantis, a high-throughput 4D microscope that integrates two complementary, gentle, live-cell imaging technologies: remote-refocus label-free microscopy and oblique light-sheet fluorescence microscopy. Additionally, we report shrimPy, an open-source software for high-throughput imaging, deconvolution, and single-cell phenotyping of 4D data. Using Mantis and shrimPy, we achieved high-content correlative imaging of molecular dynamics and the physical architecture of 20 cell lines every 15 minutes over 7.5 hours. This platform also facilitated detailed measurements of the impacts of viral infection on the architecture of host cells and host proteins. The Mantis platform can enable high-throughput profiling of intracellular dynamics, long-term imaging and analysis of cellular responses to perturbations, and live-cell optical screens to dissect gene regulatory networks.

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.

FAT10 mediates the effect of TNF-α in inducing chromosomal instability.

Tumor necrosis factor-alpha (TNF-α) plays important roles in chronic inflammation-associated tumorigenesis but the mechanisms involved remain poorly understood. Previously, we reported that high levels of FAT10 led to chromosomal instability that is mediated by an abbreviated mitotic phase. Here, we show that TNF-α induces FAT10 gene expression through TNF receptor 1 (TNFR1) and activates the NF-κB pathway in HCT116 and SW620 cells. TNF-α treatment also leads to an abbreviated mitotic phase that can be reversed by inhibiting FAT10 expression. This abbreviated mitotic phase is correlated with a TNF-α-induced reduction in the kinetochore localization of MAD2 during prometaphase which, again, can be reversed by inhibiting FAT10 gene expression. There is greater variability of chromosome numbers in HCT116 and SW620 cells treated with TNF-α than in untreated cells, which can be reversed by the introduction of short hairpin RNA (shRNA) against FAT10. The more stable chromosome numbers in HCT116 cells expressing FAT10 shRNA can revert to greater variability with the addition of a mutant FAT10 that is not recognized by the FAT10 shRNA. Upon TNF-α stimulation, higher cell death is observed when FAT10 expression is inhibited by shRNA. These data strongly suggest that FAT10 plays an important role in mediating the function of TNF-α during tumorigenesis by inducing cell cycle deregulation and chromosomal instability, and by inhibiting apoptosis.

Superresolution by image scanning microscopy using pixel reassignment.

The effect of detector array size on resolution and signal collection efficiency of image scanning microscopy based on pixel reassignment is studied. It is shown how the method can also be employed if there is a Stokes shift in fluorescence emission wavelength. With no Stokes shift, the width of the point spread function can be sharpened by a factor of 1.53, and its peak intensity increased by a factor of 1.84.

MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

Three-level filter for increased depth of focus and Bessel beam generation.

A novel axially-symmetric filter for increasing focal depth and generating an approximation to a Bessel beam is proposed. It consists of an array of rings of strength -1,0 and 1. The design is based on an analytic solution, and combines high resolution in the transverse direction with good efficiency. One presented design increases the depth of focus compared with a standard lens by more than 30 times, with a very flat axial intensity distribution over this range. Effects of discretization are discussed. Various different approaches to increasing depth of focus are compared, to put the new design into perspective.

Correlative imaging of the spatio-angular dynamics of biological systems with multimodal instant polarization microscope.

The spatial and angular organization of biological macromolecules is a key determinant, as well as informative readout, of their function. Correlative imaging of the dynamic spatio-angular architecture of cells and organelles is valuable, but remains challenging with current methods. Correlative imaging of spatio-angular dynamics requires fast polarization-, depth-, and wavelength-diverse measurement of intrinsic optical properties and fluorescent labels. We report a multimodal instant polarization microscope (miPolScope) that combines a broadband polarization-resolved detector, automation, and reconstruction algorithms to enable label-free imaging of phase, retardance, and orientation, multiplexed with fluorescence imaging of concentration, anisotropy, and orientation of molecules at diffraction-limited resolution and high speed. miPolScope enabled multimodal imaging of myofibril architecture and contractile activity of beating cardiomyocytes, cell and organelle architecture of live HEK293T and U2OS cells, and density and anisotropy of white and grey matter of mouse brain tissue across the visible spectrum. We anticipate these developments in joint quantitative imaging of density and anisotropy to enable new studies in tissue pathology, mechanobiology, and imaging-based screens.