Mechanically sheared axially swept light-sheet microscopy.

We present a mechanically sheared image acquisition format for upright and open-top light-sheet microscopes that automatically places data in its proper spatial context. This approach, which reduces computational post-processing and eliminates unnecessary interpolation or duplication of the data, is demonstrated on an upright variant of axially swept light-sheet microscopy (ASLM) that achieves a field of view, measuring 774 × 435 microns, that is 3.2-fold larger than previous models and a raw and isotropic resolution of ∼460 nm. Combined, we demonstrate the power of this approach by imaging sub-diffraction beads, cleared biological tissues, and expanded specimens.

Compression-dependent microtubule reinforcement enables cells to navigate confined environments.

Cells migrating through complex three-dimensional environments experience considerable physical challenges, including tensile stress and compression. To move, cells need to resist these forces while also squeezing the large nucleus through confined spaces. This requires highly coordinated cortical contractility. Microtubules can both resist compressive forces and sequester key actomyosin regulators to ensure appropriate activation of contractile forces. Yet, how these two roles are integrated to achieve nuclear transmigration in three dimensions is largely unknown. Here, we demonstrate that compression triggers reinforcement of a dedicated microtubule structure at the rear of the nucleus by the mechanoresponsive recruitment of cytoplasmic linker-associated proteins, which dynamically strengthens and repairs the lattice. These reinforced microtubules form the mechanostat: an adaptive feedback mechanism that allows the cell to both withstand compressive force and spatiotemporally organize contractility signalling pathways. The microtubule mechanostat facilitates nuclear positioning and coordinates force production to enable the cell to pass through constrictions. Disruption of the mechanostat imbalances cortical contractility, stalling migration and ultimately resulting in catastrophic cell rupture. Our findings reveal a role for microtubules as cellular sensors that detect and respond to compressive forces, enabling movement and ensuring survival in mechanically demanding environments.

Shifts in keratin isoform expression activate motility signals during wound healing.

Keratin intermediate filaments confer structural stability to epithelial tissues, but the reason this simple mechanical function requires a protein family with 54 isoforms is not understood. During skin wound healing, a shift in keratin isoform expression alters the composition of keratin filaments. If and how this change modulates cellular functions that support epidermal remodeling remains unclear. We report an unexpected effect of keratin isoform variation on kinase signal transduction. Increased expression of wound-associated keratin 6A, but not of steady-state keratin 5, potentiated keratinocyte migration and wound closure without compromising mechanical stability by activating myosin motors to increase contractile force generation. These results substantially expand the functional repertoire of intermediate filaments from their canonical role as mechanical scaffolds to include roles as isoform-tuned signaling scaffolds that organize signal transduction cascades in space and time to influence epithelial cell state.

Proteolysis-free amoeboid migration of melanoma cells through crowded environments via bleb-driven worrying.

In crowded microenvironments, migrating cells must find or make a path. Amoeboid cells are thought to find a path by deforming their bodies to squeeze through tight spaces. Yet, some amoeboid cells seem to maintain a near-spherical morphology as they move. To examine how they do so, we visualized amoeboid human melanoma cells in dense environments and found that they carve tunnels via bleb-driven degradation of extracellular matrix components without the need for proteolytic degradation. Interactions between adhesions and collagen at the cell front induce a signaling cascade that promotes bleb enlargement via branched actin polymerization. Large blebs abrade collagen, creating feedback between extracellular matrix structure, cell morphology, and polarization that enables both path generation and persistent movement.

A general algorithm for consensus 3D cell segmentation from 2D segmented stacks.

Cell segmentation is the fundamental task. Only by segmenting, can we define the quantitative spatial unit for collecting measurements to draw biological conclusions. Deep learning has revolutionized 2D cell segmentation, enabling generalized solutions across cell types and imaging modalities. This has been driven by the ease of scaling up image acquisition, annotation and computation. However 3D cell segmentation, which requires dense annotation of 2D slices still poses significant challenges. Labelling every cell in every 2D slice is prohibitive. Moreover it is ambiguous, necessitating cross-referencing with other orthoviews. Lastly, there is limited ability to unambiguously record and visualize 1000's of annotated cells. Here we develop a theory and toolbox, u-Segment3D for 2D-to-3D segmentation, compatible with any 2D segmentation method. Given optimal 2D segmentations, u-Segment3D generates the optimal 3D segmentation without data training, as demonstrated on 11 real life datasets, >70,000 cells, spanning single cells, cell aggregates and tissue.

Adaptive Optics in an Oblique Plane Microscope.

Adaptive optics (AO) can restore diffraction limited performance when imaging beyond superficial cell layers in vivo and in vitro, and as such is of interest for advanced 3D microscopy methods such as light-sheet fluorescence microscopy (LSFM). In a typical LSFM system, the illumination and detection paths are separate and subject to different optical aberrations. To achieve optimal microscope performance, it is necessary to sense and correct these aberrations in both light paths, resulting in a complex microscope system. Here, we show that in an oblique plane microscope (OPM), a type of LSFM with a single primary objective lens, the same deformable mirror can correct both the illumination and fluorescence detection. Besides reducing the complexity, we show that AO in OPM also restores the relative alignment of the light-sheet and focal plane, and that a projection imaging mode can stabilize and improve the wavefront correction in a sensorless AO format. We demonstrate OPM with AO on fluorescent nanospheres and by imaging the vasculature and cancer cells in zebrafish embryos embedded in a glass capillary, restoring diffraction limited resolution and improving the signal strength twofold.

Mechanically Sheared Axially Swept Light-Sheet Microscopy.

We present a mechanically sheared image acquisition format for upright and open-top light-sheet microscopes that automatically places data in its proper spatial context. This approach, which reduces computational post-processing and eliminates unnecessary interpolation or duplication of the data, is demonstrated on an upright variant of Axially Swept Light-Sheet Microscopy (ASLM) that achieves a field of view, measuring 774 x 435 microns, that is 3.2-fold larger than previous models and a raw and isotropic resolution of ∼420 nm. Combined, we demonstrate the power of this approach by imaging sub-diffraction beads, cleared biological tissues, and expanded specimens.

navigate: an open-source platform for smart light-sheet microscopy.

navigate is a turnkey, open-source software solution designed to enhance light-sheet fluorescence microscopy (LSFM) by integrating smart microscopy techniques into a user-friendly framework. It enables automated, intelligent imaging with a Python-based control system that supports GUI-reconfigurable acquisition routines and the integration of diverse hardware sets. As a comprehensive package, navigate democratizes access to advanced LSFM capabilities, facilitating the development and implementation of smart microscopy workflows without requiring deep programming knowledge or specialized expertise in light-sheet microscopy.

u-track3D: Measuring, navigating, and validating dense particle trajectories in three dimensions.

We describe u-track3D, a software package that extends the versatile u-track framework established in 2D to address the specific challenges of 3D particle tracking. First, we present the performance of the new package in quantifying a variety of intracellular dynamics imaged by multiple 3D microcopy platforms and on the standard 3D test dataset of the particle tracking challenge. These analyses indicate that u-track3D presents a tracking solution that is competitive to both conventional and deep-learning-based approaches. We then present the concept of dynamic region of interest (dynROI), which allows an experimenter to interact with dynamic 3D processes in 2D views amenable to visual inspection. Third, we present an estimator of trackability that automatically defines a score for every trajectory, thereby overcoming the challenges of trajectory validation by visual inspection. With these combined strategies, u-track3D provides a complete framework for unbiased studies of molecular processes in complex volumetric sequences.

Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro or in cells that overexpress protein, the physiological relevance of LLPS is unclear. PERIOD proteins are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Here we show that when transgene was stably expressed, PER2 formed nuclear phosphorylation-dependent LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins is a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian studies.

A comprehensive model of Drosophila epithelium reveals the role of embryo geometry and cell topology in mechanical responses.

In order to understand morphogenesis, it is necessary to know the material properties or forces shaping the living tissue. In spite of this need, very few in vivo measurements are currently available. Here, using the early Drosophila embryo as a model, we describe a novel cantilever-based technique which allows for the simultaneous quantification of applied force and tissue displacement in a living embryo. By analyzing data from a series of experiments in which embryonic epithelium is subjected to developmentally relevant perturbations, we conclude that the response to applied force is adiabatic and is dominated by elastic forces and geometric constraints, or system size effects. Crucially, computational modeling of the experimental data indicated that the apical surface of the epithelium must be softer than the basal surface, a result which we confirmed experimentally. Further, we used the combination of experimental data and comprehensive computational model to estimate the elastic modulus of the apical surface and set a lower bound on the elastic modulus of the basal surface. More generally, our investigations revealed important general features that we believe should be more widely addressed when quantitatively modeling tissue mechanics in any system. Specifically, different compartments of the same cell can have very different mechanical properties; when they do, they can contribute differently to different mechanical stimuli and cannot be merely averaged together. Additionally, tissue geometry can play a substantial role in mechanical response, and cannot be neglected.

Keratin isoform shifts modulate motility signals during wound healing.

Keratin intermediate filaments form strong mechanical scaffolds that confer structural stability to epithelial tissues, but the reason this function requires a protein family with fifty-four isoforms is not understood. During skin wound healing, a shift in keratin isoform expression alters the composition of keratin filaments. How this change modulates cellular function to support epidermal remodeling remains unclear. We report an unexpected effect of keratin isoform variation on kinase signal transduction. Increased expression of wound-associated keratin 6A, but not of steady-state keratin 5, potentiated keratinocyte migration and wound closure without compromising epidermal stability by activating myosin motors. This pathway depended on isoform-specific interaction between intrinsically disordered keratin head domains and non-filamentous vimentin shuttling myosin-activating kinases. These results substantially expand the functional repertoire of intermediate filaments from their canonical role as mechanical scaffolds to include roles as signaling scaffolds that spatiotemporally organize signal transduction cascades depending on isoform composition.

Blebs promote cell survival by assembling oncogenic signalling hubs.

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

Discoidin domain receptor 2 regulates aberrant mesenchymal lineage cell fate and matrix organization.

Extracellular matrix (ECM) interactions regulate both the cell transcriptome and proteome, thereby determining cell fate. Traumatic heterotopic ossification (HO) is a disorder characterized by aberrant mesenchymal lineage (MLin) cell differentiation, forming bone within soft tissues of the musculoskeletal system following traumatic injury. Recent work has shown that HO is influenced by ECM-MLin cell receptor signaling, but how ECM binding affects cellular outcomes remains unclear. Using time course transcriptomic and proteomic analyses, we identified discoidin domain receptor 2 (DDR2), a cell surface receptor for fibrillar collagen, as a key MLin cell regulator in HO formation. Inhibition of DDR2 signaling, through either constitutive or conditional Ddr2 deletion or pharmaceutical inhibition, reduced HO formation in mice. Mechanistically, DDR2 perturbation alters focal adhesion orientation and subsequent matrix organization, modulating Focal Adhesion Kinase (FAK) and Yes1 Associated Transcriptional Regulator and WW Domain Containing Transcription Regulator 1 (YAP/TAZ)-mediated MLin cell signaling. Hence, ECM-DDR2 interactions are critical in driving HO and could serve as a previously unknown therapeutic target for treating this disease process.

Resolution doubling in light-sheet microscopy via oblique plane structured illumination.

Structured illumination microscopy (SIM) doubles the spatial resolution of a fluorescence microscope without requiring high laser powers or specialized fluorophores. However, the excitation of out-of-focus fluorescence can accelerate photobleaching and phototoxicity. In contrast, light-sheet fluorescence microscopy (LSFM) largely avoids exciting out-of-focus fluorescence, thereby enabling volumetric imaging with low photobleaching and intrinsic optical sectioning. Combining SIM with LSFM would enable gentle three-dimensional (3D) imaging at doubled resolution. However, multiple orientations of the illumination pattern, which are needed for isotropic resolution doubling in SIM, are challenging to implement in a light-sheet format. Here we show that multidirectional structured illumination can be implemented in oblique plane microscopy, an LSFM technique that uses a single objective for excitation and detection, in a straightforward manner. We demonstrate isotropic lateral resolution below 150 nm, combined with lower phototoxicity compared to traditional SIM systems and volumetric acquisition speed exceeding 1 Hz.

Isotropic imaging across spatial scales with axially swept light-sheet microscopy.

Light-sheet fluorescence microscopy is a rapidly growing technique that has gained tremendous popularity in the life sciences owing to its high-spatiotemporal resolution and gentle, non-phototoxic illumination. In this protocol, we provide detailed directions for the assembly and operation of a versatile light-sheet fluorescence microscopy variant, referred to as axially swept light-sheet microscopy (ASLM), that delivers an unparalleled combination of field of view, optical resolution and optical sectioning. To democratize ASLM, we provide an overview of its working principle and applications to biological imaging, as well as pragmatic tips for the assembly, alignment and control of its optical systems. Furthermore, we provide detailed part lists and schematics for several variants of ASLM that together can resolve molecular detail in chemically expanded samples, subcellular organization in living cells or the anatomical composition of chemically cleared intact organisms. We also provide software for instrument control and discuss how users can tune imaging parameters to accommodate diverse sample types. Thus, this protocol will serve not only as a guide for both introductory and advanced users adopting ASLM, but as a useful resource for any individual interested in deploying custom imaging technology. We expect that building an ASLM will take ~1-2 months, depending on the experience of the instrument builder and the version of the instrument.