A spatial human thymus cell atlas mapped to a continuous tissue axis.

T cells develop from circulating precursors, which enter the thymus and migrate throughout specialised sub-compartments to support maturation and selection. This process starts already in early fetal development and is highly active until the involution of the thymus in adolescence. To map the micro-anatomical underpinnings of this process in pre- vs. post-natal states, we undertook a spatially resolved analysis and established a new quantitative morphological framework for the thymus, the Cortico-Medullary Axis. Using this axis in conjunction with the curation of a multimodal single-cell, spatial transcriptomics and high-resolution multiplex imaging atlas, we show that canonical thymocyte trajectories and thymic epithelial cells are highly organised and fully established by post-conception week 12, pinpoint TEC progenitor states, find that TEC subsets and peripheral tissue genes are associated with Hassall's Corpuscles and uncover divergence in the pace and drivers of medullary entry between CD4 vs. CD8 T cell lineages. These findings are complemented with a holistic toolkit for spatial analysis and annotation, providing a basis for a detailed understanding of T lymphocyte development.

Simulating structurally variable nuclear pore complexes for microscopy.

MOTIVATION: The nuclear pore complex (NPC) is the only passageway for macromolecules between nucleus and cytoplasm, and an important reference standard in microscopy: it is massive and stereotypically arranged. The average architecture of NPC proteins has been resolved with pseudoatomic precision, however observed NPC heterogeneities evidence a high degree of divergence from this average. Single-molecule localization microscopy (SMLM) images NPCs at protein-level resolution, whereupon image analysis software studies NPC variability. However, the true picture of this variability is unknown. In quantitative image analysis experiments, it is thus difficult to distinguish intrinsically high SMLM noise from variability of the underlying structure. RESULTS: We introduce CIR4MICS ('ceramics', Configurable, Irregular Rings FOR MICroscopy Simulations), a pipeline that synthesizes ground truth datasets of structurally variable NPCs based on architectural models of the true NPC. Users can select one or more N- or C-terminally tagged NPC proteins, and simulate a wide range of geometric variations. We also represent the NPC as a spring-model such that arbitrary deforming forces, of user-defined magnitudes, simulate irregularly shaped variations. Further, we provide annotated reference datasets of simulated human NPCs, which facilitate a side-by-side comparison with real data. To demonstrate, we synthetically replicate a geometric analysis of real NPC radii and reveal that a range of simulated variability parameters can lead to observed results. Our simulator is therefore valuable to test the capabilities of image analysis methods, as well as to inform experimentalists about the requirements of hypothesis-driven imaging studies. AVAILABILITY AND IMPLEMENTATION: Code: https://github.com/uhlmanngroup/cir4mics. Simulated data: BioStudies S-BSST1058.

Embryo-uterine interaction coordinates mouse embryogenesis during implantation.

Embryo implantation into the uterus marks a key transition in mammalian development. In mice, implantation is mediated by the trophoblast and is accompanied by a morphological transition from the blastocyst to the egg cylinder. However, the roles of trophoblast-uterine interactions in embryo morphogenesis during implantation are poorly understood due to inaccessibility in utero and the remaining challenges to recapitulate it ex vivo from the blastocyst. Here, we engineer a uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo and reveal essential roles of the physical embryo-uterine interaction. We demonstrate that adhesion between the trophoblast and the uterine matrix is required for in utero-like transition of the blastocyst to the egg cylinder. Modeling the implanting embryo as a wetting droplet links embryo shape dynamics to the underlying changes in trophoblast adhesion and suggests that the adhesion-mediated tension release facilitates egg cylinder formation. Light-sheet live imaging and the experimental control of the engineered uterine geometry and trophoblast velocity uncovers the coordination between trophoblast motility and embryo growth, where the trophoblast delineates space for embryo morphogenesis.

Made to measure: An introduction to quantifying microscopy data in the life sciences.

Images are at the core of most modern biological experiments and are used as a major source of quantitative information. Numerous algorithms are available to process images and make them more amenable to be measured. Yet the nature of the quantitative output that is useful for a given biological experiment is uniquely dependent upon the question being investigated. Here, we discuss the 3 main types of information that can be extracted from microscopy data: intensity, morphology, and object counts or categorical labels. For each, we describe where they come from, how they can be measured, and what may affect the relevance of these measurements in downstream data analysis. Acknowledging that what makes a measurement 'good' is ultimately down to the biological question being investigated, this review aims at providing readers with a toolkit to challenge how they quantify their own data and be critical of conclusions drawn from quantitative bioimage analysis experiments.

A biologist's guide to planning and performing quantitative bioimaging experiments.

Technological advancements in biology and microscopy have empowered a transition from bioimaging as an observational method to a quantitative one. However, as biologists are adopting quantitative bioimaging and these experiments become more complex, researchers need additional expertise to carry out this work in a rigorous and reproducible manner. This Essay provides a navigational guide for experimental biologists to aid understanding of quantitative bioimaging from sample preparation through to image acquisition, image analysis, and data interpretation. We discuss the interconnectedness of these steps, and for each, we provide general recommendations, key questions to consider, and links to high-quality open-access resources for further learning. This synthesis of information will empower biologists to plan and execute rigorous quantitative bioimaging experiments efficiently.

MorphoFeatures for unsupervised exploration of cell types, tissues, and organs in volume electron microscopy.

Electron microscopy (EM) provides a uniquely detailed view of cellular morphology, including organelles and fine subcellular ultrastructure. While the acquisition and (semi-)automatic segmentation of multicellular EM volumes are now becoming routine, large-scale analysis remains severely limited by the lack of generally applicable pipelines for automatic extraction of comprehensive morphological descriptors. Here, we present a novel unsupervised method for learning cellular morphology features directly from 3D EM data: a neural network delivers a representation of cells by shape and ultrastructure. Applied to the full volume of an entire three-segmented worm of the annelid Platynereis dumerilii, it yields a visually consistent grouping of cells supported by specific gene expression profiles. Integration of features across spatial neighbours can retrieve tissues and organs, revealing, for example, a detailed organisation of the animal foregut. We envision that the unbiased nature of the proposed morphological descriptors will enable rapid exploration of very different biological questions in large EM volumes, greatly increasing the impact of these invaluable, but costly resources.

Steer'n'Detect: fast 2D template detection with accurate orientation estimation.

MOTIVATION: Rotated template matching is an efficient and versatile algorithm to analyze microscopy images, as it automates the detection of stereotypical structures, such as organelles that can appear at any orientation. Its performance however quickly degrades in noisy image data. RESULTS: We introduce Steer'n'Detect, an ImageJ plugin implementing a recently published algorithm to detect patterns of interest at any orientation with high accuracy from a single template in 2D images. Steer'n'Detect provides a faster and more robust substitute to template matching. By adapting to the statistics of the image background, it guarantees accurate results even in the presence of noise. The plugin comes with an intuitive user interface facilitating results analysis and further post-processing. AVAILABILITY AND IMPLEMENTATION: https://github.com/Biomedical-Imaging-Group/Steer-n-Detect. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Deep learning for bioimage analysis in developmental biology.

Deep learning has transformed the way large and complex image datasets can be processed, reshaping what is possible in bioimage analysis. As the complexity and size of bioimage data continues to grow, this new analysis paradigm is becoming increasingly ubiquitous. In this Review, we begin by introducing the concepts needed for beginners to understand deep learning. We then review how deep learning has impacted bioimage analysis and explore the open-source resources available to integrate it into a research project. Finally, we discuss the future of deep learning applied to cell and developmental biology. We analyze how state-of-the-art methodologies have the potential to transform our understanding of biological systems through new image-based analysis and modelling that integrate multimodal inputs in space and time.

Principled Design and Implementation of Steerable Detectors.

We provide a complete pipeline for the detection of patterns of interest in an image. In our approach, the patterns are assumed to be adequately modeled by a known template, and are located at unknown positions and orientations that we aim at retrieving. We propose a continuous-domain additive image model, where the analyzed image is the sum of the patterns to localize and a background with self-similar isotropic power-spectrum. We are then able to compute the optimal filter fulfilling the SNR criterion based on one single template and background pair: it strongly responds to the template while being optimally decoupled from the background model. In addition, we constrain our filter to be steerable, which allows for a fast template detection together with orientation estimation. In practice, the implementation requires to discretize a continuous-domain formulation on polar grids, which is performed using quadratic radial B-splines. We demonstrate the practical usefulness of our method on a variety of template approximation and pattern detection experiments. We show that the detection performance drastically improves when we exploit the statistics of the background via its power-spectrum decay, which we refer to as spectral-shaping. The proposed scheme outperforms state-of-the-art steerable methods by up to 50% of absolute detection performance.

Support and approximation properties of Hermite splines.

In this paper, we formally investigate two mathematical aspects of Hermite splines that are relevant to practical applications. We first demonstrate that Hermite splines are maximally localized, in the sense that the size of their support is minimal among pairs of functions with identical reproduction properties. Then, we precisely quantify the approximation power of Hermite splines for the reconstruction of functions and their derivatives. It is known that the Hermite and B-spline approximation schemes have the same approximation order. More precisely, their approximation error vanishes as O ( T 4 ) when the step size T goes to zero. In this work, we show that they actually have the same asymptotic approximation error constants, too. Therefore, they have identical asymptotic approximation properties. Hermite splines combine optimal localization and excellent approximation power, while retaining interpolation properties and closed-form expression, in contrast to existing similar functions. These findings shed a new light on the convenience of Hermite splines in the context of computer graphics and geometrical design.

Supervised learning to quantify amyloidosis in whole brains of an Alzheimer's disease mouse model acquired with optical projection tomography.

Alzheimer's disease (AD) is characterized by amyloidosis of brain tissues. This phenomenon is studied with genetically-modified mouse models. We propose a method to quantify amyloidosis in whole 5xFAD mouse brains, a model of AD. We use optical projection tomography (OPT) and a random forest voxel classifier to segment and measure amyloid plaques. We validate our method in a preliminary cross-sectional study, where we measure 6136 ± 1637, 8477 ± 3438, and 17267 ± 4241 plaques (AVG ± SD) at 11, 17, and 31 weeks. Overall, this method can be used in the evaluation of new treatments against AD.

Multimodal imaging and high-throughput image-processing for drug screening on living organisms on-chip.

A major step for the validation of medical drugs is the screening on whole organisms, which gives the systemic information that is missing when using cellular models. Caenorhabditis elegans is a soil worm that catches the interest of researchers who study systemic physiopathology (e.g., metabolic and neurodegenerative diseases) because: (1) its large genetic homology with humans supports translational analysis; (2) worms are much easier to handle and grow in large amounts compared with rodents, for which (3) the costs and (4) the ethical concerns are substantial. Here, we demonstrate how multimodal optical imaging on such an organism can provide high-content information relevant to the drug development pipeline (e.g., mode-of-action identification, dose-response analysis), especially when combined with on-chip multiplexing capability. After designing a microfluidic array to select small separated populations of C. elegans, we combine fluorescence and bright-field imaging along with high-throughput feature recognition and signal detection to enable the identification of the mode-of-action of an antibiotic. For this purpose, we use a genetically encoded fluorescence reporter of mitochondrial stress, which we studied in living specimens during their entire development. Furthermore, we demonstrate real-time, very large field-of-view capability on multiplexed motility assays for the assessment of the dose-response relation of an anesthetic.

DiversePathsJ: diverse shortest paths for bioimage analysis.

Motivation: We introduce a formulation for the general task of finding diverse shortest paths between two end-points. Our approach is not linked to a specific biological problem and can be applied to a large variety of images thanks to its generic implementation as a user-friendly ImageJ/Fiji plugin. It relies on the introduction of additional layers in a Viterbi path graph, which requires slight modifications to the standard Viterbi algorithm rules. This layered graph construction allows for the specification of various constraints imposing diversity between solutions. Results: The software allows obtaining a collection of diverse shortest paths under some user-defined constraints through a convenient and user-friendly interface. It can be used alone or be integrated into larger image analysis pipelines. Availability and implementation: http://bigwww.epfl.ch/algorithms/diversepathsj. Contact: michael.unser@epfl.ch or fred.hamprecht@iwr.uni-heidelberg.de. Supplementary information: Supplementary data are available at Bioinformatics online.

FlyLimbTracker: An active contour based approach for leg segment tracking in unmarked, freely behaving Drosophila.

Understanding the biological underpinnings of movement and action requires the development of tools for quantitative measurements of animal behavior. Drosophila melanogaster provides an ideal model for developing such tools: the fly has unparalleled genetic accessibility and depends on a relatively compact nervous system to generate sophisticated limbed behaviors including walking, reaching, grooming, courtship, and boxing. Here we describe a method that uses active contours to semi-automatically track body and leg segments from video image sequences of unmarked, freely behaving D. melanogaster. We show that this approach yields a more than 6-fold reduction in user intervention when compared with fully manual annotation and can be used to annotate videos with low spatial or temporal resolution for a variety of locomotor and grooming behaviors. FlyLimbTracker, the software implementation of this method, is open-source and our approach is generalizable. This opens up the possibility of tracking leg movements in other species by modifications of underlying active contour models.

Multiresolution Subdivision Snakes.

We present a new family of snakes that satisfy the property of multiresolution by exploiting subdivision schemes. We show in a generic way how to construct such snakes based on an admissible subdivision mask. We derive the necessary energy formulations and provide the formulas for their efficient computation. Depending on the choice of the mask, such models have the ability to reproduce trigonometric or polynomial curves. They can also be designed to be interpolating, a property that is useful in user-interactive applications. We provide explicit examples of subdivision snakes and illustrate their use for the segmentation of bioimages. We show that they are robust in the presence of noise and provide a multiresolution algorithm to enlarge their basin of attraction, which decreases their dependence on initialization compared to singleresolution snakes. We show the advantages of the proposed model in terms of computation and segmentation of structures with different sizes.

Hermite Snakes With Control of Tangents.

We introduce a new model of parametric contours defined in a continuous fashion. Our curve model relies on Hermite spline interpolation and can easily generate curves with sharp discontinuities; it also grants direct access to the tangent at each location. With these two features, the Hermite snake distinguishes itself from classical spline-snake models and allows one to address certain bioimaging problems in a more efficient way. More precisely, the Hermite snake construction allows introducing sharp corners in the snake curve and designing directional energy functionals relying on local orientation information in the input image. Using the formalism of spline theory, the model is shown to meet practical requirements such as invariance to affine transformations and good approximation properties. Finally, the dependence on initial conditions and the robustness to the noise is studied on synthetic data in order to validate our Hermite snake model, and its usefulness is illustrated on real biological images acquired using brightfield, phase-contrast, differential-interference-contrast, and scanning-electron microscopy.

Maximally Localized Radial Profiles for Tight Steerable Wavelet Frames.

A crucial component of steerable wavelets is the radial profile of the generating function in the frequency domain. In this paper, we present an infinite-dimensional optimization scheme that helps us find the optimal profile for a given criterion over the space of tight frames. We consider two classes of criteria that measure the localization of the wavelet. The first class specifies the spatial localization of the wavelet profile, and the second that of the resulting wavelet coefficients. From these metrics and the proposed algorithm, we construct tight wavelet frames that are optimally localized and provide their analytical expression. In particular, one of the considered criterion helps us finding back the popular Simoncelli wavelet profile. Finally, the investigation of local orientation estimation, image reconstruction from detected contours in the wavelet domain, and denoising indicate that optimizing wavelet localization improves the performance of steerable wavelets, since our new wavelets outperform the traditional ones.

CP-CHARM: segmentation-free image classification made accessible.

BACKGROUND: Automated classification using machine learning often relies on features derived from segmenting individual objects, which can be difficult to automate. WND-CHARM is a previously developed classification algorithm in which features are computed on the whole image, thereby avoiding the need for segmentation. The algorithm obtained encouraging results but requires considerable computational expertise to execute. Furthermore, some benchmark sets have been shown to be subject to confounding artifacts that overestimate classification accuracy. RESULTS: We developed CP-CHARM, a user-friendly image-based classification algorithm inspired by WND-CHARM in (i) its ability to capture a wide variety of morphological aspects of the image, and (ii) the absence of requirement for segmentation. In order to make such an image-based classification method easily accessible to the biological research community, CP-CHARM relies on the widely-used open-source image analysis software CellProfiler for feature extraction. To validate our method, we reproduced WND-CHARM's results and ensured that CP-CHARM obtained comparable performance. We then successfully applied our approach on cell-based assay data and on tissue images. We designed these new training and test sets to reduce the effect of batch-related artifacts. CONCLUSIONS: The proposed method preserves the strengths of WND-CHARM - it extracts a wide variety of morphological features directly on whole images thereby avoiding the need for cell segmentation, but additionally, it makes the methods easily accessible for researchers without computational expertise by implementing them as a CellProfiler pipeline. It has been demonstrated to perform well on a wide range of bioimage classification problems, including on new datasets that have been carefully selected and annotated to minimize batch effects. This provides for the first time a realistic and reliable assessment of the whole image classification strategy.