Vladimír Ulman

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.

Bioinformatic and image analyses of the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID during apoptosis in human cells

We studied the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID in silico using three prediction tools and in living cells using both single-cell colocalization image analysis and nuclear translocation analysis. We confirmed the mitochondrial localization of endonuclease G and AIF by prediction analysis and by single-cell colocalization image analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various organelles. The highest concentration of AMID was observed associated with the Golgi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity using manual tracking analysis. Bioinformatic analysis also detected nuclear localization signals in endonuclease G and AIF, but not in AMID. A novel analysis of time-lapse fluorescence image data during staurosporine-induced apoptosis revealed nuclear translocation only for endonuclease G and AIF.

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.

Generation of synthetic image datasets for time-lapse fluorescence microscopy

In the field of biomedical image analysis, motion tracking and segmentation algorithms are important tools for time-resolved analysis of cell characteristics, events, and tracking. There are many algorithms in everyday use. Nevertheless, most of them is not properly validated as the ground truth (GT), which is a very important tool for the verification of image processing algorithms, is not naturally available. Many algorithms in this field of study are, therefore, validated only manually by an human expert. This is usually difficult, cumbersome and time consuming task, especially when single 3D image or even 3D image sequence is considered. In this paper, we have proposed a technique that generates time-lapse sequences of fully 3D synthetic image datasets. It includes generating shape, structure, and also motion of selected biological objects. The corresponding GT data is generated as well. The technique is focused on the generation of synthetic objects at various scales. Such datasets can be then processed by selected segmentation or motion tracking algorithms. The results can be compared with the GT and the quality of the applied algorithm can be measured.

Prediction of localization and interactions of apoptotic proteins

During apoptosis several mitochondrial proteins are released. Some of them participate in caspase-independent nuclear DNA degradation, especially apoptosis-inducing factor (AIF) and endonuclease G (endoG). Another interesting protein, which was expected to act similarly as AIF due to the high sequence homology with AIF is AIF-homologous mitochondrion-associated inducer of death (AMID). We studied the structure, cellular localization, and interactions of several proteins in silico and also in cells using fluorescent microscopy. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the lipid membranes. Bioinformatic predictions were conducted to analyze the interactions of the studied proteins with each other and with other possible partners. We conducted molecular modeling of proteins with unknown 3D structures. These models were then refined by MolProbity server and employed in molecular docking simulations of interactions. Our results show data acquired using a combination of modern in silico methods and image analysis to understand the localization, interactions and functions of proteins AMID, AIF, endonuclease G, and other apoptosis-related proteins.

Towards a Realistic Distribution of Cells in Synthetically Generated 3D Cell Populations

In fluorescence microscopy, the proper evaluation of image segmentation algorithms is still an open problem. In the field of cell segmentation, such evaluation can be seen as a study of the given algorithm how well it can discover individual cells as a function of the number of them in an image (size of cell population), their mutual positions (density of cell clusters), and the level of noise. Principally, there are two approaches to the evaluation. One approach requires real input images and an expert that verifies the segmentation results. This is, however, expert dependent and, namely when handling 3D data, very tedious. The second approach uses synthetic images with ground truth data to which the segmentation result is compared objectively. In this paper, we propose a new method for generating synthetic 3D images showing naturally distributed cell populations attached to microscope slide. Cell count and clustering probability are user parameters of the method.

MitoGen: A Framework for Generating 3D Synthetic Time-Lapse Sequences of Cell Populations in Fluorescence Microscopy

The proper analysis of biological microscopy images is an important and complex task. Therefore, it requires verification of all steps involved in the process, including image segmentation and tracking algorithms. It is generally better to verify algorithms with computer-generated ground truth datasets, which, compared to manually annotated data, nowadays have reached high quality and can be produced in large quantities even for 3D time-lapse image sequences. Here, we propose a novel framework, called MitoGen, which is capable of generating ground truth datasets with fully 3D time-lapse sequences of synthetic fluorescence-stained cell populations. MitoGen shows biologically justified cell motility, shape and texture changes as well as cell divisions. Standard fluorescence microscopy phenomena such as photobleaching, blur with real point spread function (PSF), and several types of noise, are simulated to obtain realistic images. The MitoGen framework is scalable in both space and time. MitoGen generates visually plausible data that shows good agreement with real data in terms of image descriptors and mean square displacement (MSD) trajectory analysis. Additionally, it is also shown in this paper that four publicly available segmentation and tracking algorithms exhibit similar performance on both real and MitoGen-generated data. The implementation of MitoGen is freely available.

Pseudo-real image sequence generator for optical flow computations

The availability of ground-truth flow field is crucial for quantitative evaluation of any optical flow computation method. The fidelity of test data is also important when artificially generated. Therefore, we generated an artificial flow field together with an artificial image sequence based on real-world sample image. The presented framework benefits of a two-layered approach in which user-selected foreground was locally moved and inserted into an artificially generated background. The background is visually similar to input sample image while the foreground is extracted from original and so is the same. The framework is capable of generating 2D and 3D image sequences of arbitrary length. Several examples of the version tuned to simulate real fluorescent microscope images are presented. We also provide a brief discussion.

Virtual cell imaging: A review on simulation methods employed in image cytometry.

The simulations of cells and microscope images thereof have been used to facilitate the development, selection, and validation of image analysis algorithms employed in cytometry as well as for modeling and understanding cell structure and dynamics beyond what is visible in the eyepiece. The simulation approaches vary from simple parametric models of specific cell components—especially shapes of cells and cell nuclei—to learning‐based synthesis and multi‐stage simulation models for complex scenes that simultaneously visualize multiple object types and incorporate various properties of the imaged objects and laws of image formation. This review covers advances in artificial digital cell generation at scales ranging from particles up to tissue synthesis and microscope image simulation methods, provides examples of the use of simulated images for various purposes ranging from subcellular object detection to cell tracking, and discusses how such simulators have been validated. Finally, the future possibilities and limitations of simulation‐based validation are considered.

On proper simulation of chromatin structure in static images as well as in time-lapse sequences in fluorescence microscopy

In fluorescence microscopy, where the benchmark datasets for validating the various image analysis methods are difficult to obtain, a great demand is either for manually annotated real image data or for realistic computer generated ones. In the last two decades, the latter case has become more and more accessible due to an increasing computer capabilities. However, the development of elaborate models, especially in the field of fluorescence microscopy imaging, is less progressive. In this paper, we propose a novel approach, based on well established concepts, to properly imitate the structure of chromatin inside the interphase cell nucleus as well as its dynamics. The performance of the approach was quantitatively evaluated against the real data. The results show that the produced images are sufficiently plausible and visually resemble their real counter parts, both for fixed and living cells.