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.

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.

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.

Surface-guided computing to analyze subcellular morphology and membrane-associated signals in 3D.

Signal transduction and cell function are governed by the spatiotemporal organization of membrane-associated molecules. Despite significant advances in visualizing molecular distributions by 3D light microscopy, cell biologists still have limited quantitative understanding of the processes implicated in the regulation of molecular signals at the whole cell scale. In particular, complex and transient cell surface morphologies challenge the complete sampling of cell geometry, membrane-associated molecular concentration and activity and the computing of meaningful parameters such as the cofluctuation between morphology and signals. Here, we introduce u-Unwrap3D, a framework to remap arbitrarily complex 3D cell surfaces and membrane-associated signals into equivalent lower dimensional representations. The mappings are bidirectional, allowing the application of image processing operations in the data representation best suited for the task and to subsequently present the results in any of the other representations, including the original 3D cell surface. Leveraging this surface-guided computing paradigm, we track segmented surface motifs in 2D to quantify the recruitment of Septin polymers by blebbing events; we quantify actin enrichment in peripheral ruffles; and we measure the speed of ruffle movement along topographically complex cell surfaces. Thus, u-Unwrap3D provides access to spatiotemporal analyses of cell biological parameters on unconstrained 3D surface geometries and signals.

Surface-guided computing to analyze subcellular morphology and membrane-associated signals in 3D.

Signal transduction and cell function are governed by the spatiotemporal organization of membrane-associated molecules. Despite significant advances in visualizing molecular distributions by 3D light microscopy, cell biologists still have limited quantitative understanding of the processes implicated in the regulation of molecular signals at the whole cell scale. In particular, complex and transient cell surface morphologies challenge the complete sampling of cell geometry, membrane-associated molecular concentration and activity and the computing of meaningful parameters such as the cofluctuation between morphology and signals. Here, we introduce u-Unwrap3D, a framework to remap arbitrarily complex 3D cell surfaces and membrane-associated signals into equivalent lower dimensional representations. The mappings are bidirectional, allowing the application of image processing operations in the data representation best suited for the task and to subsequently present the results in any of the other representations, including the original 3D cell surface. Leveraging this surface-guided computing paradigm, we track segmented surface motifs in 2D to quantify the recruitment of Septin polymers by blebbing events; we quantify actin enrichment in peripheral ruffles; and we measure the speed of ruffle movement along topographically complex cell surfaces. Thus, u-Unwrap3D provides access to spatiotemporal analyses of cell biological parameters on unconstrained 3D surface geometries and signals.

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.

Influence of chronic obstructive pulmonary disease on long-term hospitalization and mortality in patients with heart failure with reduced ejection fraction.

BACKGROUND: Heart failure with reduced ejection fraction (HFrEF) can coexist with chronic obstructive pulmonary disease (COPD), which complicates the clinical situation and worsens quality of life. The study used standard diagnostic criteria for detecting COPD in hospitalized HFrEF patients and to survey the influence of other comorbidities and medications on the long-term outcomes of HFrEF + COPD patients. METHODS: We retrospectively recruited patients hospitalized due to HFrEF in a tertiary medical center and examined and followed up clinical outcomes, including length of hospital stay, mortality, and readmission episodes, for a 5-year period. Risk factors for mortality were analyzed using multivariate analysis. RESULTS: Of the 118 hospitalized HFrEF study participants, 68 had concurrent COPD whereas 50 did not. There was a significant increase in the male predominance, smoking history, higher hemoglobin level and increased length of hospital stay in the HF + COPD group than in the HF-only group. Lower left ventricular ejection fraction was found in the HF and COPD comorbidity group. In multivariate analysis, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker (ACEI/ARB) use independently associated with a beneficial effect on survival in HF patients with COPD. Oral corticosteroid uses and stroke as a comorbidity were independently associated with a shorter time to the first readmission episode. CONCLUSION: In HFrEF patients, COPD was associated with a prolonged length of hospital stay. ACEI/ARB use might relate to a beneficial effect on survival in HF patients with COPD. The use of maintenance oral corticosteroid in patients with both HF and COPD should be crucially evaluated to determine the clinical benefit and disadvantages.

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.

Increasing the field-of-view in oblique plane microscopy via optical tiling.

Fast volumetric imaging of large fluorescent samples with high-resolution is required for many biological applications. Oblique plane microscopy (OPM) provides high spatiotemporal resolution, but the field of view is typically limited by its optical train and the pixel number of the camera. Mechanically scanning the sample or decreasing the overall magnification of the imaging system can partially address this challenge, albeit by reducing the volumetric imaging speed or spatial resolution, respectively. Here, we introduce a novel dual-axis scan unit for OPM that facilitates rapid and high-resolution volumetric imaging throughout a volume of 800 × 500 × 200 microns. This enables us to perform volumetric imaging of cell monolayers, spheroids and zebrafish embryos with subcellular resolution. Furthermore, we combined this microscope with a multi-perspective projection imaging technique that increases the volumetric interrogation rate to more than 10 Hz. This allows us to rapidly probe a large field of view in a dimensionality reduced format, identify features of interest, and volumetrically image these regions with high spatiotemporal resolution.

Real-time multi-angle projection imaging of biological dynamics.

We introduce a cost-effective and easily implementable scan unit that converts any camera-based microscope with optical sectioning capability into a multi-angle projection imaging system. Projection imaging reduces data overhead and accelerates imaging by a factor of >100, while also allowing users to readily view biological phenomena of interest from multiple perspectives on the fly. By rapidly interrogating the sample from just two perspectives, our method also enables real-time stereoscopic imaging and three-dimensional particle localization. We demonstrate projection imaging with spinning disk confocal, lattice light-sheet, multidirectional illumination light-sheet and oblique plane microscopes on specimens that range from organelles in single cells to the vasculature of a zebrafish embryo. Furthermore, we leverage our projection method to rapidly image cancer cell morphodynamics and calcium signaling in cultured neurons at rates up to 119 Hz as well as to simultaneously image orthogonal views of a beating embryonic zebrafish heart.

A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics.

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

Converting lateral scanning into axial focusing to speed up three-dimensional microscopy.

In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10-100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution.

Systematic and quantitative comparison of lattice and Gaussian light-sheets.

The axial resolving power of a light-sheet microscope is determined by the thickness of the illumination beam and the numerical aperture of its detection optics. Bessel-beam based optical lattices have generated significant interest owing to their reportedly narrow beam waist and propagation-invariant characteristics. Yet, despite their significant use in lattice light-sheet microscopy and recent incorporation into commercialized systems, there are very few quantitative reports on their physical properties and how they compare to standard Gaussian illumination beams. Here, we measure the beam properties in the transmission of dithered square lattices, which is the most commonly used variant of lattice light-sheet microscopy, and Gaussian-based light-sheets. After a systematic analysis, we find that square lattices are very similar to Gaussian-based light-sheets in terms of thickness, confocal parameter, propagation length and overall imaging performance.

Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments.

BACKGROUND: Every biological experiment requires a choice of throughput balanced against physiological relevance. Most primary drug screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput. METHODS: Here we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput. RESULTS: We demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations. CONCLUSION: In summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.

Universal light-sheet generation with field synthesis.

We introduce field synthesis, a theorem and method that can be used to synthesize any scanned or dithered light sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. Compared to LLSM, this user-friendly and modular approach offers a simplified optical design, higher light throughput and simultaneous multicolor illumination. Further, field synthesis achieves lower rates of photobleaching than light sheets generated by lateral beam scanning.

High-resolution light-scattering imaging with two-dimensional hexagonal illumination patterns: system implementation and image reconstruction formulations.

Structured illumination microscopy (SIM) was recently adapted to coherent imaging, named structured oblique-illumination microscopy (SOIM), to improve the contrast and resolution of a light-scattering image. Herein, we present high-resolution laterally isotropic SOIM imaging with 2D hexagonal illuminations. The SOIM is implemented in a SIM fluorescence system based on a spatial-light modulator (SLM). We design an SLM pattern to generate diffraction beams at 0° and ± 60.3° simultaneously to form a 2D hexagonal illumination, and undertake calculations to obtain optimal SLM shifts at 19 phases to yield a reconstructed image correctly. Beams of linear and circular polarizations are used to show the effect of polarization on the resolution improvement. We derive the distributions of the electric field of the resultant hexagonal patterns and work out the formulations of the corresponding coherent-scattering imaging for image reconstruction. The reconstructed images of gold nanoparticles (100 nm) confirm the two-fold improvement of resolution and reveal the effect of polarization on resolving adjacent nanoparticles. To demonstrate biological applications, we present the cellular structures of a label-free fixed HeLa cell with improved contrast and resolution. This work enables one to perform high-resolution dual-mode - fluorescence and light-scattering - imaging in a system, and is expected to broaden the applications of SOIM.