Martin Maška

Objective comparison of particle tracking methods

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Because manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized an open competition in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to notable practical conclusions for users and developers.

A Benchmark for Comparison of Cell Tracking Algorithms

Motivation: Automatic tracking of cells in multidimensional time-lapse fluorescence microscopy is an important task in many biomedical applications. A novel framework for objective evaluation of cell tracking algorithms has been established under the auspices of the IEEE International Symposium on Biomedical Imaging 2013 Cell Tracking Challenge. In this article, we present the logistics, datasets, methods and results of the challenge and lay down the principles for future uses of this benchmark. Results: The main contributions of the challenge include the creation of a comprehensive video dataset repository and the definition of objective measures for comparison and ranking of the algorithms. With this benchmark, six algorithms covering a variety of segmentation and tracking paradigms have been compared and ranked based on their performance on both synthetic and real datasets. Given the diversity of the datasets, we do not declare a single winner of the challenge. Instead, we present and discuss the results for each individual dataset separately. Availability and implementation: The challenge Web site (http://www.codesolorzano.com/celltrackingchallenge) provides access to the training and competition datasets, along with the ground truth of the training videos. It also provides access to Windows and Linux executable files of the evaluation software and most of the algorithms that competed in the challenge. Contact: codesolorzano@unav.es Supplementary information: Supplementary data are available at Bioinformatics online.

Segmentation of Touching Cell Nuclei Using a Two-Stage Graph Cut Model

Methods based on combinatorial graph cut algorithms received a lot of attention in the recent years for their robustness as well as reasonable computational demands. These methods are built upon an underlying Maximum a Posteriori estimation of Markov Random Fields and are suitable to solve accurately many different problems in image analysis, including image segmentation. In this paper we present a two-stage graph cut based model for segmentation of touching cell nuclei in fluorescence microscopy images. In the first stage voxels with very high probability of being foreground or background are found and separated by a boundary with a minimal geodesic length. In the second stage the obtained clusters are split into isolated cells by combining image gradient information and incorporated a priori knowledge about the shape of the nuclei. Moreover, these two qualities can be easily balanced using a single user parameter. Preliminary tests on real data show promising results of the method.

An objective comparison of cell-tracking algorithms

We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays todays state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods. We also analyzed the performance of all of the algorithms in terms of biological measures and practical usability. Although some methods scored high in all technical aspects, none obtained fully correct solutions. We found that methods that either take prior information into account using learning strategies or analyze cells in a global spatiotemporal video context performed better than other methods under the segmentation and tracking scenarios included in the challenge.

On simulating 3D fluorescent microscope images

In recent years many various biomedical image segmentation methods have appeared. Though typically presented to be successful the majority of them was not properly tested against ground truth images. The obvious way of testing the quality of new segmentation was based on visual inspection by a specialist in the given field. The novel 3D biomedical image data simulator is presented in this paper. It offers the results of high quality. The comparison of generated synthetic data is compared against real image data using standard similarity techniques.

Characterization of three-dimensional cancer cell migration in mixed collagen-Matrigel scaffolds using microfluidics and image analysis

Microfluidic devices are becoming mainstream tools to recapitulate in vitro the behavior of cells and tissues. In this study, we use microfluidic devices filled with hydrogels of mixed collagen-Matrigel composition to study the migration of lung cancer cells under different cancer invasion microenvironments. We present the design of the microfluidic device, characterize the hydrogels morphologically and mechanically and use quantitative image analysis to measure the migration of H1299 lung adenocarcinoma cancer cells in different experimental conditions. Our results show the plasticity of lung cancer cell migration, which turns from mesenchymal in collagen only matrices, to lobopodial in collagen-Matrigel matrices that approximate the interface between a disrupted basement membrane and the underlying connective tissue. Our quantification of migration speed confirms a biphasic role of Matrigel. At low concentration, Matrigel facilitates migration, most probably by providing a supportive and growth factor retaining environment. At high concentration, Matrigel slows down migration, possibly due excessive attachment. Finally, we show that antibody-based integrin blockade promotes a change in migration phenotype from mesenchymal or lobopodial to amoeboid and analyze the effect of this change in migration dynamics, in regards to the structure of the matrix. In summary, we describe and characterize a robust microfluidic platform and a set of software tools that can be used to study lung cancer cell migration under different microenvironments and experimental conditions. This platform could be used in future studies, thus benefitting from the advantages introduced by microfluidic devices: precise control of the environment, excellent optical properties, parallelization for high throughput studies and efficient use of therapeutic drugs.

Cell tracking accuracy measurement based on comparison of acyclic oriented graphs

Tracking motile cells in time-lapse series is challenging and is required in many biomedical applications. Cell tracks can be mathematically represented as acyclic oriented graphs. Their vertices describe the spatio-temporal locations of individual cells, whereas the edges represent temporal relationships between them. Such a representation maintains the knowledge of all important cellular events within a captured field of view, such as migration, division, death, and transit through the field of view. The increasing number of cell tracking algorithms calls for comparison of their performance. However, the lack of a standardized cell tracking accuracy measure makes the comparison impracticable. This paper defines and evaluates an accuracy measure for objective and systematic benchmarking of cell tracking algorithms. The measure assumes the existence of a ground-truth reference, and assesses how difficult it is to transform a computed graph into the reference one. The difficulty is measured as a weighted sum of the lowest number of graph operations, such as split, delete, and add a vertex and delete, add, and alter the semantics of an edge, needed to make the graphs identical. The measure behavior is extensively analyzed based on the tracking results provided by the participants of the first Cell Tracking Challenge hosted by the 2013 IEEE International Symposium on Biomedical Imaging. We demonstrate the robustness and stability of the measure against small changes in the choice of weights for diverse cell tracking algorithms and fluorescence microscopy datasets. As the measure penalizes all possible errors in the tracking results and is easy to compute, it may especially help developers and analysts to tune their algorithms according to their needs.

Smooth Chan-Vese segmentation via graph cuts

The graph cut framework presents an efficient method for approximating the minimum of the popular Chan-Vese functional for image segmentation. However, a fundamental drawback of graph cuts is a need for a dense neighbourhood system in order to avoid geometric artefacts and jagged boundaries. The increasing connectivity leads to excessive memory consumption and burdens the efficiency of the method. In this paper, we address the issue by introducing a two-stage connectivity scaling approach. First, coarse segmentation is calculated using a sparse neighbourhood over the whole image. In the second stage, the segmentation is refined by employing a dense neighbourhood in a narrow band around the boundary from the first stage. We demonstrate that this method fits well with the Chan-Vese functional and yields smooth boundaries without increasing the computational demands significantly. Moreover, under specific conditions, the construction has no negative effect on the optimality of the solution.

Acquiarium: Free software for the acquisition and analysis of 3D images of cells in fluorescence microscopy

This paper describes free software (called Acquiarium, http://cbia.fi.muni.cz/acquiarium.html) for carrying out the common pipeline of many spatial cell studies using fluorescence microscopy. It addresses image capture, raw image correction, image segmentation, the quantification and spatial arrangement of segmented objects, volume rendering, and statistical evaluation. The software is designed for the easy processing of a collection of many 3D images. It can be used for the analysis of a collection of 2D images or time lapse series of 2D or 3D images as well. It has a modular design and is extensible via plug-ins. The paper gives an overview of the software and its key design ideas.

The role of chromatin condensation during granulopoiesis in the regulation of gene cluster expression

Changes in nuclear architecture play an important role in the regulation of gene expression. The importance of epigenetic changes is observed during granulopoiesis, when changes in the nuclear architecture are considered a major factor that influences the downregulation of genes. We aimed to assess the influence of chromatin condensation on the regulation of gene expression during granulopoiesis. Based on a previously published microarray analysis, we chose loci with different levels of transcriptional activity during granulopoiesis. Fluorescent in situ hybridisation (FISH) and immunofluorescent labelling of RNA polymerase II were used to determine the relationship between the transcriptional activity of gene clusters and their localisation within areas with different levels of chromatin condensation. Although active loci were positioned outside of areas of condensed chromatin, downregulation of genes during granulopoiesis was not accompanied by a shift of the downregulated loci to condensed areas. Only the beta-globin cluster was subjected to chromatin condensation and localised to condensed areas. Our results indicate that granulopoiesis is accompanied by a non-random, tissue-specific pattern of chromatin condensation. Furthermore, we observed that the decrease in the quantity of RNA polymerase II correlates with the differentiation process and likely acts in synergy with chromatin condensation to downregulate total gene expression.