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 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.

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.

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.

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.

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.

Projective light-sheet microscopy with flexible parameter selection.

Projection imaging accelerates volumetric interrogation in fluorescence microscopy, but for multi-cellular samples, the resulting images may lack contrast, as many structures and haze are summed up. Here, we demonstrate rapid projective light-sheet imaging with parameter selection (props) of imaging depth, position and viewing angle. This allows us to selectively image different sub-volumes of a sample, rapidly switch between them and exclude background fluorescence. Here we demonstrate the power of props by functional imaging within distinct regions of the zebrafish brain, monitoring calcium firing inside muscle cells of moving Drosophila larvae, super-resolution imaging of selected cell layers, and by optically unwrapping the curved surface of a Drosophila embryo. We anticipate that props will accelerate volumetric interrogation, ranging from subcellular to mesoscopic scales.

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.

The Vps13-like protein BLTP2 is pro-survival and regulates phosphatidylethanolamine levels in the plasma membrane to maintain its fluidity and function.

Lipid transport proteins (LTPs) facilitate nonvesicular lipid exchange between cellular compartments and have critical roles in lipid homeostasis1. A new family of bridge-like LTPs (BLTPs) is thought to form lipid-transporting conduits between organelles2. One, BLTP2, is conserved across species but its function is not known. Here, we show that BLTP2 and its homolog directly regulate plasma membrane (PM) fluidity by increasing the phosphatidylethanolamine (PE) level in the PM. BLTP2 localizes to endoplasmic reticulum (ER)-PM contact sites34, 5, suggesting it transports PE from the ER to the PM. We find BLTP2 works in parallel with another pathway that regulates intracellular PE distribution and PM fluidity6, 7. BLTP2 expression correlates with breast cancer aggressiveness8-10. We found BLTP2 facilitates growth of a human cancer cell line and sustains its aggressiveness in an in vivo model of metastasis, suggesting maintenance of PM fluidity by BLTP2 may be critical for tumorigenesis in humans.

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.

Mesoscopic Oblique Plane Microscopy via Light-sheet Mirroring.

Understanding the intricate interplay and inter-connectivity of biological processes across an entire organism is important in various fields of biology, including cardiovascular research, neuroscience, and developmental biology. Here, we present a mesoscopic oblique plane microscope (OPM) that enables whole organism imaging with high speed and subcellular resolution. A microprism underneath the sample enhances the axial resolution and optical sectioning through total internal reflection of the light-sheet. Through rapid refocusing of the light-sheet, the imaging depth is extended up to threefold while keeping the axial resolution constant. Using low magnification objectives with a large field of view, we realize mesoscopic imaging over a volume of 3.7×1.5×1 mm3 with ~2.3 microns lateral and ~9.2 microns axial resolution. Applying the mesoscopic OPM, we demonstrate in vivo and in toto whole organism imaging of the zebrafish vasculature and its endothelial nuclei, and blood flow dynamics at 12 Hz acquisition rate, resulting in a quantitative map of blood flow across the entire organism.

Projective oblique plane structured illumination microscopy.

Structured illumination microscopy (SIM) can double the spatial resolution of a fluorescence microscope and video rate live cell imaging in a two-dimensional format has been demonstrated. However, rapid implementations of 2D SIM typically only cover a narrow slice of the sample immediately at the coverslip, with most of the cellular volume out of reach. Here we implement oblique plane structured illumination microscopy (OPSIM) in a projection format to rapidly image an entire cell in a 2D SIM framework. As no mechanical scanning of the sample or objective is involved, this technique has the potential for rapid projection imaging with doubled resolution. We characterize the spatial resolution with fluorescent nanospheres, compare projection and 3D imaging using OPSIM and image mitochondria and ER dynamics across an entire cell at up to 2.7 Hz. To our knowledge, this represents the fastest whole cell SIM imaging to date.

Light-sheets and smart microscopy, an exciting future is dawning.

Light-sheet fluorescence microscopy has transformed our ability to visualize and quantitatively measure biological processes rapidly and over long time periods. In this review, we discuss current and future developments in light-sheet fluorescence microscopy that we expect to further expand its capabilities. This includes smart and adaptive imaging schemes to overcome traditional imaging trade-offs, i.e., spatiotemporal resolution, field of view and sample health. In smart microscopy, a microscope will autonomously decide where, when, what and how to image. We further assess how image restoration techniques provide avenues to overcome these tradeoffs and how "open top" light-sheet microscopes may enable multi-modal imaging with high throughput. As such, we predict that light-sheet microscopy will fulfill an important role in biomedical and clinical imaging in the future.

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.

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.

The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of ß-catenin.

The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated ß-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of ß-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of ß-catenin. This article has an associated First Person interview with the first authors of the paper.

In vivo 3D profiling of site-specific human cancer cell morphotypes in zebrafish.

Tissue microenvironments affect the functional states of cancer cells, but determining these influences in vivo has remained a challenge. We present a quantitative high-resolution imaging assay of single cancer cells in zebrafish xenografts to probe functional adaptation to variable cell-extrinsic cues and molecular interventions. Using cell morphology as a surrogate readout of cell functional states, we examine environmental influences on the morphotype distribution of Ewing Sarcoma, a pediatric cancer associated with the oncogene EWSR1-FLI1 and whose plasticity is thought to determine disease outcome through non-genomic mechanisms. Computer vision analysis reveals systematic shifts in the distribution of 3D morphotypes as a function of cell type and seeding site, as well as tissue-specific cellular organizations that recapitulate those observed in human tumors. Reduced expression of the EWSR1-FLI1 protein product causes a shift to more protrusive cells and decreased tissue specificity of the morphotype distribution. Overall, this work establishes a framework for a statistically robust study of cancer cell plasticity in diverse tissue microenvironments.

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.