Multi-sample SPIM image acquisition, processing and analysis of vascular growth in zebrafish.

To quantitatively understand biological processes that occur over many hours or days, it is desirable to image multiple samples simultaneously, and automatically process and analyse the resulting datasets. Here, we present a complete multi-sample preparation, imaging, processing and analysis workflow to determine the development of the vascular volume in zebrafish. Up to five live embryos were mounted and imaged simultaneously over several days using selective plane illumination microscopy (SPIM). The resulting large imagery dataset of several terabytes was processed in an automated manner on a high-performance computer cluster and segmented using a novel segmentation approach that uses images of red blood cells as training data. This analysis yielded a precise quantification of growth characteristics of the whole vascular network, head vasculature and tail vasculature over development. Our multi-sample platform demonstrates effective upgrades to conventional single-sample imaging platforms and paves the way for diverse quantitative long-term imaging studies.

Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish.

We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures "kugeln", after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch-dependent NO-containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.

Cell-intrinsic adaptation of lipid composition to local crowding drives social behaviour.

Cells sense the context in which they grow to adapt their phenotype and allow multicellular patterning by mechanisms of autocrine and paracrine signalling. However, patterns also form in cell populations exposed to the same signalling molecules and substratum, which often correlate with specific features of the population context of single cells, such as local cell crowding. Here we reveal a cell-intrinsic molecular mechanism that allows multicellular patterning without requiring specific communication between cells. It acts by sensing the local crowding of a single cell through its ability to spread and activate focal adhesion kinase (FAK, also known as PTK2), resulting in adaptation of genes controlling membrane homeostasis. In cells experiencing low crowding, FAK suppresses transcription of the ABC transporter A1 (ABCA1) by inhibiting FOXO3 and TAL1. Agent-based computational modelling and experimental confirmation identified membrane-based signalling and feedback control as crucial for the emergence of population patterns of ABCA1 expression, which adapts membrane lipid composition to cell crowding and affects multiple signalling activities, including the suppression of ABCA1 expression itself. The simple design of this cell-intrinsic system and its broad impact on the signalling state of mammalian single cells suggests a fundamental role for a tunable membrane lipid composition in collective cell behaviour.

Endothelial cell self-fusion during vascular pruning.

During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell-cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies.

Singularity Containers Improve Reproducibility and Ease of Use in Computational Image Analysis Workflows.

Reproducing computational workflows in image analysis and microscopy can be a daunting task due to different software versions and dependencies. This is especially true for users with little specific knowledge of scientific computation. To overcome these challenges, we introduce Singularity containers as a useful tool to run and share image analysis workflows among many users, even years later after establishing them. Unfortunately, containers are rarely used so far in the image analysis field. To address this lack of use, we provide a detailed step-by-step protocol to package a state-of-the-art segmentation algorithm into a container on a local Windows machine to run the container on a high-performance cluster computer.

U-Hack Med Gap Year-A Virtual Undergraduate Internship Program in Computer-Assisted Healthcare and Biomedical Research.

The COVID-19 healthcare crisis dramatically changed educational opportunities for undergraduate students. To overcome the lack of exposure to lab research and provide an alternative to cancelled classes and online lectures, the Lyda Hill Department of Bioinformatics at UT Southwestern Medical Center established an innovative, fully remote and paid "U-Hack Med Gap Year" internship program. At the core of the internship program were dedicated biomedical research projects spanning nine months in fields as diverse as computational microscopy, bioimage analysis, genome sequence analysis and establishment of a surgical skill analysis platform. To complement the project work, a biweekly Gap Year lab meeting was devised with opportunities to develop important skills in presenting, data sharing and analysis of new research. Despite a challenging year, all selected students completed the full internship period and over 30% will continue their project remotely after the end of the program.

KRas-transformed epithelia cells invade and partially dedifferentiate by basal cell extrusion.

Metastasis is the main cause of carcinoma-related death, yet we know little about how it initiates due to our inability to visualize stochastic invasion events. Classical models suggest that cells accumulate mutations that first drive formation of a primary mass, and then downregulate epithelia-specific genes to cause invasion and metastasis. Here, using transparent zebrafish epidermis to model simple epithelia, we can directly image invasion. We find that KRas-transformation, implicated in early carcinogenesis steps, directly drives cell invasion by hijacking a process epithelia normally use to promote death-cell extrusion. Cells invading by basal cell extrusion simultaneously pinch off their apical epithelial determinants, endowing new plasticity. Following invasion, cells divide, enter the bloodstream, and differentiate into stromal, neuronal-like, and other cell types. Yet, only invading KRasV12 cells deficient in p53 survive and form internal masses. Together, we demonstrate that KRas-transformation alone causes cell invasion and partial dedifferentiation, independently of mass formation.

Fiji plugin for annotating movies with custom arrows.

Annotation of time-lapse data provides an important tool to highlight dynamic processes. Particularly, arrows, circles and arrowheads are useful to pinpoint a specific process, stationary or evolving over time. Here, we describe a user-friendly Fiji plugin to facilitate annotation of movies with arrows, arrowheads and circles. The plugin also enables saving and loading of annotated tracks.This article has an associated First Person interview with the first author of the paper.

Extended depth of focus multiphoton microscopy via incoherent pulse splitting.

We present a beam splitter mask that can be easily added to a multiphoton raster scanning microscope to extend the depth of focus five-fold at a small loss in lateral resolution. The method is designed for ultrafast laser pulses or other light-sources featuring a low coherence length. In contrast to other methods of focus extension, our approach uniquely combines low complexity, high light-throughput and multicolor capability. We characterize the point spread function in a two-photon microscope and demonstrate fluorescence imaging of GFP labeled neurons in fixed brain samples as imaged with conventional and extended depth of focus two-photon microscopy.