Jeff W. Lichtman

Zudi Lin, Donglai Wei, Jeff Lichtman et al.

We present PyTorch Connectomics (PyTC), an open-source deep-learning framework for the semantic and instance segmentation of volumetric microscopy images, built upon PyTorch. We demonstrate the effectiveness of PyTC in the field of connectomics, which aims to segment and reconstruct neurons, synapses, and other organelles like mitochondria at nanometer resolution for understanding neuronal communication, metabolism, and development in animal brains. PyTC is a scalable and flexible toolbox that tackles datasets at different scales and supports multi-task and semi-supervised learning to better exploit expensive expert annotations and the vast amount of unlabeled data during training. Those functionalities can be easily realized in PyTC by changing the configuration options without coding and adapted to other 2D and 3D segmentation tasks for different tissues and imaging modalities. Quantitatively, our framework achieves the best performance in the CREMI challenge for synaptic cleft segmentation (outperforms existing best result by relatively 6.1$\%$) and competitive performance on mitochondria and neuronal nuclei segmentation. Code and tutorials are publicly available at https://connectomics.readthedocs.io.

Abhimanyu Talwar, Zudi Lin, Donglai Wei et al.

One of the essential tasks in connectomics is the morphology analysis of neurons and organelles like mitochondria to shed light on their biological properties. However, these biological objects often have tangled parts or complex branching patterns, which make it hard to abstract, categorize, and manipulate their morphology. In this paper, we develop a novel topological nomenclature system to name these objects like the appellation for chemical compounds to promote neuroscience analysis based on their skeletal structures. We first convert the volumetric representation into the topology-preserving reduced graph to untangle the objects. Next, we develop nomenclature rules for pyramidal neurons and mitochondria from the reduced graph and finally learn the feature embedding for shape manipulation. In ablation studies, we quantitatively show that graphs generated by our proposed method align with the perception of experts. On 3D shape retrieval and decomposition tasks, we qualitatively demonstrate that the encoded topological nomenclature features achieve better results than state-of-the-art shape descriptors. To advance neuroscience, we will release a 3D segmentation dataset of mitochondria and pyramidal neurons reconstructed from a 100um cube electron microscopy volume with their reduced graph and topological nomenclature annotations. Code is publicly available at https://github.com/donglaiw/ibexHelper.

Yaron Meirovitch, Fuming Yang, Jeff Lichtman et al.

Most pruning methods remove parameters ranked by impact on loss (e.g., magnitude or gradient). We propose Budgeted Broadcast (BB), which gives each unit a local traffic budget (the product of its long-term on-rate $a_i$ and fan-out $k_i$). A constrained-entropy analysis shows that maximizing coding entropy under a global traffic budget yields a selectivity-audience balance, $\log\frac{1-a_i}{a_i}=βk_i$. BB enforces this balance with simple local actuators that prune either fan-in (to lower activity) or fan-out (to reduce broadcast). In practice, BB increases coding entropy and decorrelation and improves accuracy at matched sparsity across Transformers for ASR, ResNets for face identification, and 3D U-Nets for synapse prediction, sometimes exceeding dense baselines. On electron microscopy images, it attains state-of-the-art F1 and PR-AUC under our evaluation protocol. BB is easy to integrate and suggests a path toward learning more diverse and efficient representations.

Leslie Gu, Jason Ken Adhinarta, Mikhail Bessmeltsev et al. · harvard

Accurately segmenting 3D curvilinear structures in medical imaging remains challenging due to their complex geometry and the scarcity of diverse, large-scale datasets for algorithm development and evaluation. In this paper, we use dendritic spine segmentation as a case study and address these challenges by introducing a novel Frenet--Serret Frame-based Decomposition, which decomposes 3D curvilinear structures into a globally \( C^2 \) continuous curve that captures the overall shape, and a cylindrical primitive that encodes local geometric properties. This approach leverages Frenet--Serret Frames and arc length parameterization to preserve essential geometric features while reducing representational complexity, facilitating data-efficient learning, improved segmentation accuracy, and generalization on 3D curvilinear structures. To rigorously evaluate our method, we introduce two datasets: CurviSeg, a synthetic dataset for 3D curvilinear structure segmentation that validates our method's key properties, and DenSpineEM, a benchmark for dendritic spine segmentation, which comprises 4,476 manually annotated spines from 70 dendrites across three public electron microscopy datasets, covering multiple brain regions and species. Our experiments on DenSpineEM demonstrate exceptional cross-region and cross-species generalization: models trained on the mouse somatosensory cortex subset achieve 91.9\% Dice, maintaining strong performance in zero-shot segmentation on both mouse visual cortex (94.1\% Dice) and human frontal lobe (81.8\% Dice) subsets. Moreover, we test the generalizability of our method on the IntrA dataset, where it achieves 77.08\% Dice (5.29\% higher than prior arts) on intracranial aneurysm segmentation. These findings demonstrate the potential of our approach for accurately analyzing complex curvilinear structures across diverse medical imaging fields.

Jia Wan, Wanhua Li, Jason Ken Adhinarta et al.

While imaging techniques at macro and mesoscales have garnered substantial attention and resources, microscale Volume Electron Microscopy (vEM) imaging, capable of revealing intricate vascular details, has lacked the necessary benchmarking infrastructure. In this paper, we address a significant gap in this field of neuroimaging by introducing the first-in-class public benchmark, BvEM, designed specifically for cortical blood vessel segmentation in vEM images. Our BvEM benchmark is based on vEM image volumes from three mammals: adult mouse, macaque, and human. We standardized the resolution, addressed imaging variations, and meticulously annotated blood vessels through semi-automatic, manual, and quality control processes, ensuring high-quality 3D segmentation. Furthermore, we developed a zero-shot cortical blood vessel segmentation method named TriSAM, which leverages the powerful segmentation model SAM for 3D segmentation. To extend SAM from 2D to 3D volume segmentation, TriSAM employs a multi-seed tracking framework, leveraging the reliability of certain image planes for tracking while using others to identify potential turning points. This approach effectively achieves long-term 3D blood vessel segmentation without model training or fine-tuning. Experimental results show that TriSAM achieved superior performances on the BvEM benchmark across three species. Our dataset, code, and model are available online at \url{https://jia-wan.github.io/bvem}.

Zudi Lin, Donglai Wei, Mariela D. Petkova et al.

Segmenting 3D cell nuclei from microscopy image volumes is critical for biological and clinical analysis, enabling the study of cellular expression patterns and cell lineages. However, current datasets for neuronal nuclei usually contain volumes smaller than $10^{\text{-}3}\ mm^3$ with fewer than 500 instances per volume, unable to reveal the complexity in large brain regions and restrict the investigation of neuronal structures. In this paper, we have pushed the task forward to the sub-cubic millimeter scale and curated the NucMM dataset with two fully annotated volumes: one $0.1\ mm^3$ electron microscopy (EM) volume containing nearly the entire zebrafish brain with around 170,000 nuclei; and one $0.25\ mm^3$ micro-CT (uCT) volume containing part of a mouse visual cortex with about 7,000 nuclei. With two imaging modalities and significantly increased volume size and instance numbers, we discover a great diversity of neuronal nuclei in appearance and density, introducing new challenges to the field. We also perform a statistical analysis to illustrate those challenges quantitatively. To tackle the challenges, we propose a novel hybrid-representation learning model that combines the merits of foreground mask, contour map, and signed distance transform to produce high-quality 3D masks. The benchmark comparisons on the NucMM dataset show that our proposed method significantly outperforms state-of-the-art nuclei segmentation approaches. Code and data are available at https://connectomics-bazaar.github.io/proj/nucMM/index.html.

Donglai Wei, Kisuk Lee, Hanyu Li et al.

Electron microscopy (EM) enables the reconstruction of neural circuits at the level of individual synapses, which has been transformative for scientific discoveries. However, due to the complex morphology, an accurate reconstruction of cortical axons has become a major challenge. Worse still, there is no publicly available large-scale EM dataset from the cortex that provides dense ground truth segmentation for axons, making it difficult to develop and evaluate large-scale axon reconstruction methods. To address this, we introduce the AxonEM dataset, which consists of two 30x30x30 um^3 EM image volumes from the human and mouse cortex, respectively. We thoroughly proofread over 18,000 axon instances to provide dense 3D axon instance segmentation, enabling large-scale evaluation of axon reconstruction methods. In addition, we densely annotate nine ground truth subvolumes for training, per each data volume. With this, we reproduce two published state-of-the-art methods and provide their evaluation results as a baseline. We publicly release our code and data at https://connectomics-bazaar.github.io/proj/AxonEM/index.html to foster the development of advanced methods.

Felix Gonda, Xueying Wang, Johanna Beyer et al.

A connectivity graph of neurons at the resolution of single synapses provides scientists with a tool for understanding the nervous system in health and disease. Recent advances in automatic image segmentation and synapse prediction in electron microscopy (EM) datasets of the brain have made reconstructions of neurons possible at the nanometer scale. However, automatic segmentation sometimes struggles to segment large neurons correctly, requiring human effort to proofread its output. General proofreading involves inspecting large volumes to correct segmentation errors at the pixel level, a visually intensive and time-consuming process. This paper presents the design and implementation of an analytics framework that streamlines proofreading, focusing on connectivity-related errors. We accomplish this with automated likely-error detection and synapse clustering that drives the proofreading effort with highly interactive 3D visualizations. In particular, our strategy centers on proofreading the local circuit of a single cell to ensure a basic level of completeness. We demonstrate our framework's utility with a user study and report quantitative and subjective feedback from our users. Overall, users find the framework more efficient for proofreading, understanding evolving graphs, and sharing error correction strategies.

Toufiq Parag, Daniel Berger, Lee Kamentsky et al.

Synaptic connectivity detection is a critical task for neural reconstruction from Electron Microscopy (EM) data. Most of the existing algorithms for synapse detection do not identify the cleft location and direction of connectivity simultaneously. The few methods that computes direction along with contact location have only been demonstrated to work on either dyadic (most common in vertebrate brain) or polyadic (found in fruit fly brain) synapses, but not on both types. In this paper, we present an algorithm to automatically predict the location as well as the direction of both dyadic and polyadic synapses. The proposed algorithm first generates candidate synaptic connections from voxelwise predictions of signed proximity generated by a 3D U-net. A second 3D CNN then prunes the set of candidates to produce the final detection of cleft and connectivity orientation. Experimental results demonstrate that the proposed method outperforms the existing methods for determining synapses in both rodent and fruit fly brain.

Toufiq Parag, Fabian Tschopp, William Grisaitis et al.

The field of connectomics has recently produced neuron wiring diagrams from relatively large brain regions from multiple animals. Most of these neural reconstructions were computed from isotropic (e.g., FIBSEM) or near isotropic (e.g., SBEM) data. In spite of the remarkable progress on algorithms in recent years, automatic dense reconstruction from anisotropic data remains a challenge for the connectomics community. One significant hurdle in the segmentation of anisotropic data is the difficulty in generating a suitable initial over-segmentation. In this study, we present a segmentation method for anisotropic EM data that agglomerates a 3D over-segmentation computed from the 3D affinity prediction. A 3D U-net is trained to predict 3D affinities by the MALIS approach. Experiments on multiple datasets demonstrates the strength and robustness of the proposed method for anisotropic EM segmentation.

David Rolnick, Yaron Meirovitch, Toufiq Parag et al.

Deep learning algorithms for connectomics rely upon localized classification, rather than overall morphology. This leads to a high incidence of erroneously merged objects. Humans, by contrast, can easily detect such errors by acquiring intuition for the correct morphology of objects. Biological neurons have complicated and variable shapes, which are challenging to learn, and merge errors take a multitude of different forms. We present an algorithm, MergeNet, that shows 3D ConvNets can, in fact, detect merge errors from high-level neuronal morphology. MergeNet follows unsupervised training and operates across datasets. We demonstrate the performance of MergeNet both on a variety of connectomics data and on a dataset created from merged MNIST images.

Daniel Haehn, Verena Kaynig, James Tompkin et al.

Automatic cell image segmentation methods in connectomics produce merge and split errors, which require correction through proofreading. Previous research has identified the visual search for these errors as the bottleneck in interactive proofreading. To aid error correction, we develop two classifiers that automatically recommend candidate merges and splits to the user. These classifiers use a convolutional neural network (CNN) that has been trained with errors in automatic segmentations against expert-labeled ground truth. Our classifiers detect potentially-erroneous regions by considering a large context region around a segmentation boundary. Corrections can then be performed by a user with yes/no decisions, which reduces variation of information 7.5x faster than previous proofreading methods. We also present a fully-automatic mode that uses a probability threshold to make merge/split decisions. Extensive experiments using the automatic approach and comparing performance of novice and expert users demonstrate that our method performs favorably against state-of-the-art proofreading methods on different connectomics datasets.

Arthur W. Wetzel, Jennifer Bakal, Markus Dittrich et al.

The detailed reconstruction of neural anatomy for connectomics studies requires a combination of resolution and large three-dimensional data capture provided by serial section electron microscopy (ssEM). The convergence of high throughput ssEM imaging and improved tissue preparation methods now allows ssEM capture of complete specimen volumes up to cubic millimeter scale. The resulting multi-terabyte image sets span thousands of serial sections and must be precisely registered into coherent volumetric forms in which neural circuits can be traced and segmented. This paper introduces a Signal Whitening Fourier Transform Image Registration approach (SWiFT-IR) under development at the Pittsburgh Supercomputing Center and its use to align mouse and zebrafish brain datasets acquired using the wafer mapper ssEM imaging technology recently developed at Harvard University. Unlike other methods now used for ssEM registration, SWiFT-IR modifies its spatial frequency response during image matching to maximize a signal-to-noise measure used as its primary indicator of alignment quality. This alignment signal is more robust to rapid variations in biological content and unavoidable data distortions than either phase-only or standard Pearson correlation, thus allowing more precise alignment and statistical confidence. These improvements in turn enable an iterative registration procedure based on projections through multiple sections rather than more typical adjacent-pair matching methods. This projection approach, when coupled with known anatomical constraints and iteratively applied in a multi-resolution pyramid fashion, drives the alignment into a smooth form that properly represents complex and widely varying anatomical content such as the full cross-section zebrafish data.

Yaron Meirovitch, Alexander Matveev, Hayk Saribekyan et al.

The field of connectomics faces unprecedented "big data" challenges. To reconstruct neuronal connectivity, automated pixel-level segmentation is required for petabytes of streaming electron microscopy data. Existing algorithms provide relatively good accuracy but are unacceptably slow, and would require years to extract connectivity graphs from even a single cubic millimeter of neural tissue. Here we present a viable real-time solution, a multi-pass pipeline optimized for shared-memory multicore systems, capable of processing data at near the terabyte-per-hour pace of multi-beam electron microscopes. The pipeline makes an initial fast-pass over the data, and then makes a second slow-pass to iteratively correct errors in the output of the fast-pass. We demonstrate the accuracy of a sparse slow-pass reconstruction algorithm and suggest new methods for detecting morphological errors. Our fast-pass approach provided many algorithmic challenges, including the design and implementation of novel shallow convolutional neural nets and the parallelization of watershed and object-merging techniques. We use it to reconstruct, from image stack to skeletons, the full dataset of Kasthuri et al. (463 GB capturing 120,000 cubic microns) in a matter of hours on a single multicore machine rather than the weeks it has taken in the past on much larger distributed systems.

Felix Gonda, Verena Kaynig, Ray Thouis et al.

We present an interactive approach to train a deep neural network pixel classifier for the segmentation of neuronal structures. An interactive training scheme reduces the extremely tedious manual annotation task that is typically required for deep networks to perform well on image segmentation problems. Our proposed method employs a feedback loop that captures sparse annotations using a graphical user interface, trains a deep neural network based on recent and past annotations, and displays the prediction output to users in almost real-time. Our implementation of the algorithm also allows multiple users to provide annotations in parallel and receive feedback from the same classifier. Quick feedback on classifier performance in an interactive setting enables users to identify and label examples that are more important than others for segmentation purposes. Our experiments show that an interactively-trained pixel classifier produces better region segmentation results on Electron Microscopy (EM) images than those generated by a network of the same architecture trained offline on exhaustive ground-truth labels.

Ayushi Sinha, William Gray Roncal, Narayanan Kasthuri et al.

Accurately estimating the wiring diagram of a brain, known as a connectome, at an ultrastructure level is an open research problem. Specifically, precisely tracking neural processes is difficult, especially across many image slices. Here, we propose a novel method to automatically identify and annotate small subcellular structures present in axons, known as axoplasmic reticula, through a 3D volume of high-resolution neural electron microscopy data. Our method produces high precision annotations, which can help improve automatic segmentation by using our results as seeds for segmentation, and as cues to aid segment merging.

Ayushi Sinha, William Gray Roncal, Narayanan Kasthuri et al.

In this paper, we present a new pipeline which automatically identifies and annotates axoplasmic reticula, which are small subcellular structures present only in axons. We run our algorithm on the Kasthuri11 dataset, which was color corrected using gradient-domain techniques to adjust contrast. We use a bilateral filter to smooth out the noise in this data while preserving edges, which highlights axoplasmic reticula. These axoplasmic reticula are then annotated using a morphological region growing algorithm. Additionally, we perform Laplacian sharpening on the bilaterally filtered data to enhance edges, and repeat the morphological region growing algorithm to annotate more axoplasmic reticula. We track our annotations through the slices to improve precision, and to create long objects to aid in segment merging. This method annotates axoplasmic reticula with high precision. Our algorithm can easily be adapted to annotate axoplasmic reticula in different sets of brain data by changing a few thresholds. The contribution of this work is the introduction of a straightforward and robust pipeline which annotates axoplasmic reticula with high precision, contributing towards advancements in automatic feature annotations in neural EM data.

Verena Kaynig, Amelio Vazquez-Reina, Seymour Knowles-Barley et al.

Automated sample preparation and electron microscopy enables acquisition of very large image data sets. These technical advances are of special importance to the field of neuroanatomy, as 3D reconstructions of neuronal processes at the nm scale can provide new insight into the fine grained structure of the brain. Segmentation of large-scale electron microscopy data is the main bottleneck in the analysis of these data sets. In this paper we present a pipeline that provides state-of-the art reconstruction performance while scaling to data sets in the GB-TB range. First, we train a random forest classifier on interactive sparse user annotations. The classifier output is combined with an anisotropic smoothing prior in a Conditional Random Field framework to generate multiple segmentation hypotheses per image. These segmentations are then combined into geometrically consistent 3D objects by segmentation fusion. We provide qualitative and quantitative evaluation of the automatic segmentation and demonstrate large-scale 3D reconstructions of neuronal processes from a $\mathbf{27,000}$ $\mathbf{μm^3}$ volume of brain tissue over a cube of $\mathbf{30 \; μm}$ in each dimension corresponding to 1000 consecutive image sections. We also introduce Mojo, a proofreading tool including semi-automated correction of merge errors based on sparse user scribbles.