Tracking of plant cells in images obtained by microscope is a challenging problem due to biological phenomena such as large number of cells, non-uniform growth of different layers of the tightly packed plant cells and cell division. Moreover, images in deeper layers of the tissue being noisy and unavoidable systemic errors inherent in the imaging process further complicates the problem. In this paper, we propose a novel learning-based method that exploits the tightly packed three-dimensional cell structure of plant cells to create a three-dimensional graph in order to perform accurate cell tracking. We further propose novel algorithms for cell division detection and effective three-dimensional registration, which improve upon the state-of-the-art algorithms. We demonstrate the efficacy of our algorithm in terms of tracking accuracy and inference-time on a benchmark dataset.
In this work, we describe a method for large-scale 3D cell-tracking through a segmentation selection approach. The proposed method is effective at tracking cells across large microscopy datasets on two fronts: (i) It can solve problems containing millions of segmentation instances in terabyte-scale 3D+t datasets; (ii) It achieves competitive results with or without deep learning, which requires 3D annotated data, that is scarce in the fluorescence microscopy field. The proposed method computes cell tracks and segments using a hierarchy of segmentation hypotheses and selects disjoint segments by maximizing the overlap between adjacent frames. We show that this method achieves state-of-the-art results in 3D images from the cell tracking challenge and has a faster integer linear programming formulation. Moreover, our framework is flexible and supports segmentations from off-the-shelf cell segmentation models and can combine them into an ensemble that improves tracking. The code is available https://github.com/royerlab/ultrack.
The accurate tracking of live cells using video microscopy recordings remains a challenging task for popular state-of-the-art image processing based object tracking methods. In recent years, several existing and new applications have attempted to integrate deep-learning based frameworks for this task, but most of them still heavily rely on consecutive frame based tracking embedded in their architecture or other premises that hinder generalized learning. To address this issue, we aimed to develop a new deep-learning based tracking method that relies solely on the assumption that cells can be tracked based on their spatio-temporal neighborhood, without restricting it to consecutive frames. The proposed method has the additional benefit that the motion patterns of the cells can be learned completely by the predictor without any prior assumptions, and it has the potential to handle a large number of video frames with heavy artifacts. The efficacy of the proposed method is demonstrated through biologically motivated validation strategies and compared against multiple state-of-the-art cell tracking methods.
Data-driven cell tracking and segmentation methods in biomedical imaging require diverse and information-rich training data. In cases where the number of training samples is limited, synthetic computer-generated data sets can be used to improve these methods. This requires the synthesis of cell shapes as well as corresponding microscopy images using generative models. To synthesize realistic living cell shapes, the shape representation used by the generative model should be able to accurately represent fine details and changes in topology, which are common in cells. These requirements are not met by 3D voxel masks, which are restricted in resolution, and polygon meshes, which do not easily model processes like cell growth and mitosis. In this work, we propose to represent living cell shapes as level sets of signed distance functions (SDFs) which are estimated by neural networks. We optimize a fully-connected neural network to provide an implicit representation of the SDF value at any point in a 3D+time domain, conditioned on a learned latent code that is disentangled from the rotation of the cell shape. We demonstrate the effectiveness of this approach on cells that exhibit rapid deformations (Platynereis dumerilii), cells that grow and divide (C. elegans), and cells that have growing and branching filopodial protrusions (A549 human lung carcinoma cells). A quantitative evaluation using shape features and Dice similarity coefficients of real and synthetic cell shapes shows that our model can generate topologically plausible complex cell shapes in 3D+time with high similarity to real living cell shapes. Finally, we show how microscopy images of living cells that correspond to our generated cell shapes can be synthesized using an image-to-image model.
Background and objective: Cell migration is essential for many biological phenomena with direct impact on human health and disease. One conventional approach to study cell migration involves the quantitative analysis of individual cell trajectories recorded by time-lapse video microscopy. Dedicated software tools exist to assist the automated or semi-automated tracking of cells and translate these into coordinate positions along time. However, cell biologists usually bump into the difficulty of plotting and computing these data sets into biologically meaningful figures and metrics. Methods: This report describes MigraR, an intuitive graphical user interface executed from the RStudioTM (via the R package Shiny), which greatly simplifies the task of translating coordinate positions of moving cells into measurable parameters of cell migration (velocity, straightness, and direction of movement), as well as of plotting cell trajectories and migration metrics. One innovative function of this interface is that it allows users to refine their data sets by setting limits based on time, velocity and straightness. Results: MigraR was tested on different data to assess its applicability. Intended users of MigraR are cell biologists with no prior knowledge of data analysis, seeking to accelerate the quantification and visualization of cell migration data sets delivered in the format of Excel files by available cell-tracking software. Conclusions: Through the graphics it provides, MigraR is an useful tool for the analysis of migration parameters and cellular trajectories. Since its source code is open, it can be subject of refinement by expert users to best suit the needs of other researchers. It is available at GitHub and can be easily reproduced.
The automated segmentation and tracking of macrophages during their migration are challenging tasks due to their dynamically changing shapes and motions. This paper proposes a new algorithm to achieve automatic cell tracking in time-lapse microscopy macrophage data. First, we design a segmentation method employing space-time filtering, local Otsu's thresholding, and the SUBSURF (subjective surface segmentation) method. Next, the partial trajectories for cells overlapping in the temporal direction are extracted in the segmented images. Finally, the extracted trajectories are linked by considering their direction of movement. The segmented images and the obtained trajectories from the proposed method are compared with those of the semi-automatic segmentation and manual tracking. The proposed tracking achieved 97.4% of accuracy for macrophage data under challenging situations, feeble fluorescent intensity, irregular shapes, and motion of macrophages. We expect that the automatically extracted trajectories of macrophages can provide pieces of evidence of how macrophages migrate depending on their polarization modes in the situation, such as during wound healing.
Cell tracking is an essential tool in live-cell imaging to determine single-cell features, such as division patterns or elongation rates. Unlike in common multiple object tracking, in microbial live-cell experiments cells are growing, moving, and dividing over time, to form cell colonies that are densely packed in mono-layer structures. With increasing cell numbers, following the precise cell-cell associations correctly over many generations becomes more and more challenging, due to the massively increasing number of possible associations. To tackle this challenge, we propose a fast parameter-free cell tracking approach, which consists of activity-prioritized nearest neighbor assignment of growing cells and a combinatorial solver that assigns splitting mother cells to their daughters. As input for the tracking, Omnipose is utilized for instance segmentation. Unlike conventional nearest-neighbor-based tracking approaches, the assignment steps of our proposed method are based on a Gaussian activity-based metric, predicting the cell-specific migration probability, thereby limiting the number of erroneous assignments. In addition to being a building block for cell tracking, the proposed activity map is a standalone tracking-free metric for indicating cell activity. Finally, we perform a quantitative analysis of the tracking accuracy for different frame rates, to inform life scientists about a suitable (in terms of tracking performance) choice of the frame rate for their cultivation experiments, when cell tracks are the desired key outcome.
The automated analysis of microscopy images is a challenge in the context of single-cell tracking and quantification. This work has as goals the study of the performance of deep learning for segmenting microscopy images and the improvement of the previously available pipeline for tracking single cells. Deep learning techniques, mainly convolutional neural networks, have been applied to cell segmentation problems and have shown high accuracy and fast performance. To perform the image segmentation, an analysis of hyperparameters was done in order to implement a convolutional neural network with U-Net architecture. Furthermore, different models were built in order to optimize the size of the network and the number of learnable parameters. The trained network is then used in the pipeline that localizes the traps in a microfluidic device, performs the image segmentation on trap images, and evaluates the fluorescence intensity and the area of single cells over time. The tracking of the cells during an experiment is performed by image processing algorithms, such as centroid estimation and watershed. Finally, with all improvements in the neural network to segment single cells and in the pipeline, quasi-real-time image analysis was enabled, where 6.20GB of data was processed in 4 minutes.
Tracking all nuclei of an embryo in noisy and dense fluorescence microscopy data is a challenging task. We build upon a recent method for nuclei tracking that combines weakly-supervised learning from a small set of nuclei center point annotations with an integer linear program (ILP) for optimal cell lineage extraction. Our work specifically addresses the following challenging properties of C. elegans embryo recordings: (1) Many cell divisions as compared to benchmark recordings of other organisms, and (2) the presence of polar bodies that are easily mistaken as cell nuclei. To cope with (1), we devise and incorporate a learnt cell division detector. To cope with (2), we employ a learnt polar body detector. We further propose automated ILP weights tuning via a structured SVM, alleviating the need for tedious manual set-up of a respective grid search. Our method outperforms the previous leader of the cell tracking challenge on the Fluo-N3DH-CE embryo dataset. We report a further extensive quantitative evaluation on two more C. elegans datasets. We will make these datasets public to serve as an extended benchmark for future method development. Our results suggest considerable improvements yielded by our method, especially in terms of the correctness of division event detection and the number and length of fully correct track segments. Code: https://github.com/funkelab/linajea
We share our recent findings in an attempt to train a universal segmentation network for various cell types and imaging modalities. Our method was built on the generalized U-Net architecture, which allows the evaluation of each component individually. We modified the traditional binary training targets to include three classes for direct instance segmentation. Detailed experiments were performed regarding training schemes, training settings, network backbones, and individual modules on the segmentation performance. Our proposed training scheme draws minibatches in turn from each dataset, and the gradients are accumulated before an optimization step. We found that the key to training a universal network is all-time supervision on all datasets, and it is necessary to sample each dataset in an unbiased way. Our experiments also suggest that there might exist common features to define cell boundaries across cell types and imaging modalities, which could allow application of trained models to totally unseen datasets. A few training tricks can further boost the segmentation performance, including uneven class weights in the cross-entropy loss function, well-designed learning rate scheduler, larger image crops for contextual information, and additional loss terms for unbalanced classes. We also found that segmentation performance can benefit from group normalization layer and Atrous Spatial Pyramid Pooling module, thanks to their more reliable statistics estimation and improved semantic understanding, respectively. We participated in the 6th Cell Tracking Challenge (CTC) held at IEEE International Symposium on Biomedical Imaging (ISBI) 2021 using one of the developed variants. Our method was evaluated as the best runner up during the initial submission for the primary track, and also secured the 3rd place in an additional round of competition in preparation for the summary publication.
The nematode Caenorhabditis elegans (C. elegans) is used as a model organism to better understand developmental biology and neurobiology. C. elegans features an invariant cell lineage, which has been catalogued and observed using fluorescence microscopy images. However, established methods to track cells in late-stage development fail to generalize once sporadic muscular twitching has begun. We build upon methodology which uses skin cells as fiducial markers to carry out cell tracking despite random twitching. In particular, we present a cell nucleus segmentation and tracking procedure which was integrated into a 3D rendering GUI to improve efficiency in tracking cells across late-stage development. Results on images depicting aforementioned muscle cell nuclei across three test embryos suggest the fiducial markers in conjunction with a classic tracking paradigm overcome sporadic twitching.
Methods allowing the synthesis of realistic cell shapes could help generate training data sets to improve cell tracking and segmentation in biomedical images. Deep generative models for cell shape synthesis require a light-weight and flexible representation of the cell shape. However, commonly used voxel-based representations are unsuitable for high-resolution shape synthesis, and polygon meshes have limitations when modeling topology changes such as cell growth or mitosis. In this work, we propose to use level sets of signed distance functions (SDFs) to represent cell shapes. We optimize a neural network as an implicit neural representation of the SDF value at any point in a 3D+time domain. The model is conditioned on a latent code, thus allowing the synthesis of new and unseen shape sequences. We validate our approach quantitatively and qualitatively on C. elegans cells that grow and divide, and lung cancer cells with growing complex filopodial protrusions. Our results show that shape descriptors of synthetic cells resemble those of real cells, and that our model is able to generate topologically plausible sequences of complex cell shapes in 3D+time.
A systematic analysis of the cell behavior requires automated approaches for cell segmentation and tracking. While deep learning has been successfully applied for the task of cell segmentation, there are few approaches for simultaneous cell segmentation and tracking using deep learning. Here, we present EmbedTrack, a single convolutional neural network for simultaneous cell segmentation and tracking which predicts easy to interpret embeddings. As embeddings, offsets of cell pixels to their cell center and bandwidths are learned. We benchmark our approach on nine 2D data sets from the Cell Tracking Challenge, where our approach performs on seven out of nine data sets within the top 3 contestants including three top 1 performances. The source code is publicly available at https://git.scc.kit.edu/kit-loe-ge/embedtrack.
Volumetric cell segmentation in fluorescence microscopy images is important to study a wide variety of cellular processes. Applications range from the analysis of cancer cells to behavioral studies of cells in the embryonic stage. Like in other computer vision fields, most recent methods use either large convolutional neural networks (CNNs) or vision transformer models (ViTs). Since the number of available 3D microscopy images is typically limited in applications, we take a different approach and introduce a small CNN for volumetric cell segmentation. Compared to previous CNN models for cell segmentation, our model is efficient and has an asymmetric encoder-decoder structure with very few parameters in the decoder. Training efficiency is further improved via transfer learning. In addition, we introduce Context Aware Pseudocoloring to exploit spatial context in z-direction of 3D images while performing volumetric cell segmentation slice-wise. We evaluated our method using different 3D datasets from the Cell Segmentation Benchmark of the Cell Tracking Challenge. Our segmentation method achieves top-ranking results, while our CNN model has an up to 25x lower number of parameters than other top-ranking methods. Code and pretrained models are available at: https://github.com/roydenwa/efficient-cell-seg
We present a novel graph neural network (GNN) approach for cell tracking in high-throughput microscopy videos. By modeling the entire time-lapse sequence as a direct graph where cell instances are represented by its nodes and their associations by its edges, we extract the entire set of cell trajectories by looking for the maximal paths in the graph. This is accomplished by several key contributions incorporated into an end-to-end deep learning framework. We exploit a deep metric learning algorithm to extract cell feature vectors that distinguish between instances of different biological cells and assemble same cell instances. We introduce a new GNN block type which enables a mutual update of node and edge feature vectors, thus facilitating the underlying message passing process. The message passing concept, whose extent is determined by the number of GNN blocks, is of fundamental importance as it enables the `flow' of information between nodes and edges much behind their neighbors in consecutive frames. Finally, we solve an edge classification problem and use the identified active edges to construct the cells' tracks and lineage trees. We demonstrate the strengths of the proposed cell tracking approach by applying it to 2D and 3D datasets of different cell types, imaging setups, and experimental conditions. We show that our framework outperforms current state-of-the-art methods on most of the evaluated datasets. The code is available at our repository: https://github.com/talbenha/cell-tracker-gnn.
We present a massively parallel Lagrange decomposition method for solving 0--1 integer linear programs occurring in structured prediction. We propose a new iterative update scheme for solving the Lagrangean dual and a perturbation technique for decoding primal solutions. For representing subproblems we follow Lange et al. (2021) and use binary decision diagrams (BDDs). Our primal and dual algorithms require little synchronization between subproblems and optimization over BDDs needs only elementary operations without complicated control flow. This allows us to exploit the parallelism offered by GPUs for all components of our method. We present experimental results on combinatorial problems from MAP inference for Markov Random Fields, quadratic assignment and cell tracking for developmental biology. Our highly parallel GPU implementation improves upon the running times of the algorithms from Lange et al. (2021) by up to an order of magnitude. In particular, we come close to or outperform some state-of-the-art specialized heuristics while being problem agnostic. Our implementation is available at https://github.com/LPMP/BDD.
Current methods in multiple object tracking (MOT) rely on independent object trajectories undergoing predictable motion to effectively track large numbers of objects. Adversarial conditions such as volatile object motion and imperfect detections create a challenging tracking landscape in which established methods may yield inadequate results. Multiple hypothesis hypergraph tracking (MHHT) is developed to perform MOT among interdependent objects amid noisy detections. The method extends traditional multiple hypothesis tracking (MHT) via hypergraphs to model correlated object motion, allowing for robust tracking in challenging scenarios. MHHT is applied to perform seam cell tracking during late-stage embryogenesis in embryonic C. elegans.
Matching cells over time has long been the most difficult step in cell tracking. In this paper, we approach this problem by recasting it as a classification problem. We construct a feature set for each cell, and compute a feature difference vector between a cell in the current frame and a cell in a previous frame. Then we determine whether the two cells represent the same cell over time by training decision trees as our binary classifiers. With the output of decision trees, we are able to formulate an assignment problem for our cell association task and solve it using a modified version of the Hungarian algorithm.
Recent advances in bioimaging have provided scientists a superior high spatial-temporal resolution to observe dynamics of living cells as 3D volumetric videos. Unfortunately, the 3D biomedical video analysis is lagging, impeded by resource insensitive human curation using off-the-shelf 3D analytic tools. Herein, biologists often need to discard a considerable amount of rich 3D spatial information by compromising on 2D analysis via maximum intensity projection. Recently, pixel embedding-based cell instance segmentation and tracking provided a neat and generalizable computing paradigm for understanding cellular dynamics. In this work, we propose a novel spatial-temporal voxel-embedding (VoxelEmbed) based learning method to perform simultaneous cell instance segmenting and tracking on 3D volumetric video sequences. Our contribution is in four-fold: (1) The proposed voxel embedding generalizes the pixel embedding with 3D context information; (2) Present a simple multi-stream learning approach that allows effective spatial-temporal embedding; (3) Accomplished an end-to-end framework for one-stage 3D cell instance segmentation and tracking without heavy parameter tuning; (4) The proposed 3D quantification is memory efficient via a single GPU with 12 GB memory. We evaluate our VoxelEmbed method on four 3D datasets (with different cell types) from the ISBI Cell Tracking Challenge. The proposed VoxelEmbed method achieved consistent superior overall performance (OP) on two densely annotated datasets. The performance is also competitive on two sparsely annotated cohorts with 20.6% and 2% of data-set having segmentation annotations. The results demonstrate that the VoxelEmbed method is a generalizable and memory-efficient solution.
Cell instance segmentation in fluorescence microscopy images is becoming essential for cancer dynamics and prognosis. Data extracted from cancer dynamics allows to understand and accurately model different metabolic processes such as proliferation. This enables customized and more precise cancer treatments. However, accurate cell instance segmentation, necessary for further cell tracking and behavior analysis, is still challenging in scenarios with high cell concentration and overlapping edges. Within this framework, we propose a novel cell instance segmentation approach based on the well-known U-Net architecture. To enforce the learning of morphological information per pixel, a deep distance transformer (DDT) acts as a back-bone model. The DDT output is subsequently used to train a top-model. The following top-models are considered: a three-class (\emph{e.g.,} foreground, background and cell border) U-net, and a watershed transform. The obtained results suggest a performance boost over traditional U-Net architectures. This opens an interesting research line around the idea of injecting morphological information into a fully convolutional model.