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43 Janelia Publications
Showing 31-40 of 43 resultsSerial section Microscopy is an established method for volumetric anatomy reconstruction. Section series imaged with Electron Microscopy are currently vital for the reconstruction of the synaptic connectivity of entire animal brains such as that of Drosophila melanogaster. The process of removing ultrathin layers from a solid block containing the specimen, however, is a fragile procedure and has limited precision with respect to section thickness. We have developed a method to estimate the relative z-position of each individual section as a function of signal change across the section series. First experiments show promising results on both serial section Transmission Electron Microscopy (ssTEM) data and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) series. We made our solution available as Open Source plugins for the TrakEM2 software and the ImageJ distribution Fiji.
Motivation: A significant focus of biological research is to understand the development, organization and function of tissues. A particularly productive area of study is on single layer epithelial tissues in which the adherence junctions of cells form a 2D manifold that is fluorescently labeled. Given the size of the tissue, a microscope must collect a mosaic of overlapping 3D stacks encompassing the stained surface. Downstream interpretation is greatly simplified by preprocessing such a dataset as follows: (a) extracting and mapping the stained manifold in each stack into a single 2D projection plane, (b) correcting uneven illumination artifacts, (c) stitching the mosaic planes into a single, large 2D image, and (d) adjusting the contrast. Results: We have developed PreMosa, an efficient, fully automatic pipeline to perform the four preprocessing tasks above resulting in a single 2D image of the stained manifold across which contrast is optimized and illumination is even. Notable features are as follows. First, the 2D projection step employs a specially developed algorithm that actually finds the manifold in the stack based on maximizing contrast, intensity and smoothness. Second, the projection step comes first, implying all subsequent tasks are more rapidly solved in 2D. And last, the mosaic melding employs an algorithm that globally adjusts contrasts amongst the 2D tiles so as to produce a seamless, high-contrast image. We conclude with an evaluation using ground-truth datasets and present results on datasets from Drosophila melanogaster wings and Schmidtae mediterranea ciliary components. Availability: PreMosa is available under https://cblasse.github.io/premosa. Contact: blasse@mpi-cbg.de, myers@mpi-cbg.de.
Neuronal circuit mapping using electron microscopy demands laborious proofreading or reconciliation of multiple independent reconstructions. Here, we describe new methods to apply quantitative arbor and network context to iteratively proofread and reconstruct circuits and create anatomically enriched wiring diagrams. We measured the morphological underpinnings of connectivity in new and existing reconstructions of Drosophila sensorimotor (larva) and visual (adult) systems. Synaptic inputs were preferentially located on numerous small, microtubule-free 'twigs' which branch off a single microtubule-containing 'backbone'. Omission of individual twigs accounted for 96% of errors. However, the synapses of highly connected neurons were distributed across multiple twigs. Thus, the robustness of a strong connection to detailed twig anatomy was associated with robustness to reconstruction error. By comparing iterative reconstruction to the consensus of multiple reconstructions, we show that our method overcomes the need for redundant effort through the discovery and application of relationships between cellular neuroanatomy and synaptic connectivity.
Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.
Multi-modal image registration is a challenging task that is vital to fuse complementary signals for subsequent analyses. Despite much research into cost functions addressing this challenge, there exist cases in which these are ineffective. In this work, we show that (1) this is true for the registration of in-vivo Drosophila brain volumes visualizing genetically encoded calcium indicators to an nc82 atlas and (2) that machine learning based contrast synthesis can yield improvements. More specifically, the number of subjects for which the registration outright failed was greatly reduced (from 40% to 15%) by using a synthesized image.
Many scientific software platforms provide plugin mechanisms that simplify the integration, deployment, and execution of externally developed functionality. One of the most widely used platforms in the imaging space is Fiji, a popular open-source application for scientific image analysis. Fiji incorporates and builds on the ImageJ and ImageJ2 platforms, which provide a powerful plugin architecture used by thousands of plugins to solve a wide variety of problems. This capability is a major part of Fiji's success, and it has become a widely used biological image analysis tool and a target for new functionality. However, a plugin-based software architecture cannot unify disparate platforms operating on incompatible data structures; interoperability necessitates the creation of adaptation or "bridge" layers to translate data and invoke functionality. As a result, while platforms like Fiji enable a high degree of interconnectivity and extensibility, they were not fundamentally designed to integrate across the many data types, programming languages, and architectural differences of various software help address this challenge, we present SciJava Ops, a foundational software library for expressing algorithms as plugins in a unified and extensible way. Continuing the evolution of Fiji's SciJava plugin mechanism, SciJava Ops enables users to harness algorithms from various software platforms within a central execution environment. In addition, SciJava Ops automatically adapts data into the most appropriate structure for each algorithm, allowing users to freely and transparently combine algorithms from otherwise incompatible tools. While SciJava Ops is initially distributed as a Fiji update site, the framework does not require Fiji, ImageJ, or ImageJ2, and would be suitable for integration with additional image analysis platforms.
Neural circuit reconstruction at single synapse resolution is increasingly recognized as crucially important to decipher the function of biological nervous systems. Volume electron microscopy in serial transmission or scanning mode has been demonstrated to provide the necessary resolution to segment or trace all neurites and to annotate all synaptic connections.
Automatic annotation of synaptic connections has been done successfully in near isotropic electron microscopy of vertebrate model organisms. Results on non-isotropic data in insect models, however, are not yet on par with human annotation.
We designed a new 3D-U-Net architecture to optimally represent isotropic fields of view in non-isotropic data. We used regression on a signed distance transform of manually annotated synaptic clefts of the CREMI challenge dataset to train this model and observed significant improvement over the state of the art.
We developed open source software for optimized parallel prediction on very large volumetric datasets and applied our model to predict synaptic clefts in a 50 tera-voxels dataset of the complete Drosophila brain. Our model generalizes well to areas far away from where training data was available.
Two successful approaches for the segmentation of biomedical images are (1) the selection of segment candidates from a merge-tree, and (2) the clustering of small superpixels by solving a Multi-Cut problem. In this paper, we introduce a model that unifies both approaches. Our model, the Candidate Multi-Cut (CMC), allows joint selection and clustering of segment candidates from a merge-tree. This way, we overcome the respective limitations of the individual methods: (1) the space of possible segmentations is not constrained to candidates of a merge-tree, and (2) the decision for clustering can be made on candidates larger than superpixels, using features over larger contexts. We solve the optimization problem of selecting and clustering of candidates using an integer linear program. On datasets of 2D light microscopy of cell populations and 3D electron microscopy of neurons, we show that our method generalizes well and generates more accurate segmentations than merge-tree or Multi-Cut methods alone.
Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1-86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (SPAST; SPG4), the membrane-bound GTPase atlastin-1 (ATL1; SPG3A), and the reticulon-like, microtubule-binding protein REEP1 (REEP1; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later-onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, while its binding partner Reep1 is knocked out. Atl1KI/KI/Reep1-/- mice exhibit early-onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, based on reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in Atl1KI/KI/Reep1-/- spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. Atl1KI/KI/Reep1-/- mice represent an early-onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies.