Meijering, E.

Methods for cell and particle tracking (Article)

2012-01-23T00:00:00Z

Achieving complete understanding of any living thing inevitably requires thorough analysis of both its anatomic and dynamic properties. Live-cell imaging experiments carried out to this end often produce massive amounts of time-lapse image data containing far more information than can be digested by a human observer. Computerized image analysis offers the potential to take full advantage of available data in an efficient and reproducible manner. A recurring task in many experiments is the tracking of large numbers of cells or particles and the analysis of their (morpho)dynamic behavior. In the past decade, many methods have been developed for this purpose, and software tools based on these are increasingly becoming available. Here, we survey the latest developments in this area and discuss the various computational approaches, software tools, and quantitative measures for tracking and motion analysis of cells and particles in time-lapse microscopy images.

Trans-dimensional MCMC methods for fully automatic motion analysis in tagged MRI (Article)

2011-10-11T00:00:00Z

Tagged magnetic resonance imaging (tMRI) is a well-known noninvasive method allowing quantitative analysis of regional heart dynamics. Its clinical use has so far been limited, in part due to the lack of robustness and accuracy of existing tag tracking algorithms in dealing with low (and intrinsically time-varying) image quality. In this paper, we propose a novel probabilistic method for tag tracking, implemented by means of Bayesian particle filtering and a trans-dimensional Markov chain Monte Carlo (MCMC) approach, which efficiently combines information about the imaging process and tag appearance with prior knowledge about the heart dynamics obtained by means of non-rigid image registration. Experiments using synthetic image data (with ground truth) and real data (with expert manual annotation) from preclinical (small animal) and clinical (human) studies confirm that the proposed method yields higher consistency, accuracy, and intrinsic tag reliability assessment in comparison with other frequently used tag tracking methods.

Insights into EB1 structure and the role of its c-terminal domain for discriminating microtubule tips from the lattice (Article)

2011-08-15T00:00:00Z

End-binding proteins (EBs) comprise a conserved family of microtubule plus end - tracking proteins. The concerted action of calponin homology (CH), linker, and C-terminal domains of EBs is important for their autonomous microtubule tip tracking, regulation of microtubule dynamics, and recruitment of numerous partners to microtubule ends. Here we report the detailed structural and biochemical analysis of mammalian EBs. Small-angle X-ray scattering, electron microscopy, and chemical cross-linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains, an arrangement reminiscent of that seen in other microtubule- and actin-binding proteins. Removal of the negatively charged C-terminal tail did not affect the overall conformation of EBs; however, it increased the dwell times of EBs on the microtubule lattice in microtubule tip-tracking reconstitution experiments. An even more stable association with the microtubule lattice was observed when the entire negatively charged C-terminal domain of EBs was replaced by a neutral coiled-coil motif. In contrast, the interaction of EBs with growing microtubule tips was not significantly affected by these C-terminal domain mutations. Our data indicate that long-range electrostatic repulsive interactions between the C-terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends.

Rab6, Rab8, and MICAL3 cooperate in controlling docking and fusion of exocytotic carriers (Article)

2011-06-07T00:00:00Z

Rab6 is a conserved small GTPase that localizes to the Golgi apparatus and cytoplasmic vesicles and controls transport and fusion of secretory carriers [1]. Another Rab implicated in trafficking from the trans-Golgi to the plasma membrane is Rab8 [2-5]. Here we show that Rab8A stably associates with exocytotic vesicles in a Rab6-dependent manner. Rab8A function is not needed for budding or motility of exocytotic carriers but is required for their docking and fusion. These processes also depend on the Rab6-interacting cortical factor ELKS [1], suggesting that Rab8A and ELKS act in the same pathway. We show that Rab8A and ELKS can be linked by MICAL3, a member of the MICAL family of flavoprotein monooxygenases [6]. Expression of a MICAL3 mutant with an inactive monooxygenase domain resulted in a strong accumulation of secretory vesicles that were docked at the cell cortex but failed to fuse with the plasma membrane, an effect that correlated with the strongly reduced mobility of MICAL3. We propose that the monooxygenase activity of MICAL3 is required to regulate its own turnover and the concomitant remodeling of vesicle-docking protein complexes in which it is engaged. Taken together, the results of our study illustrate cooperation of two Rab proteins in constitutive exocytosis and implicates a redox enzyme in this process.

Predictive impact of allele-matching and EBMT risk score for outcome after T-cell depleted unrelated donor transplantation in poor-risk acute leukemia and myelodysplasia (Article)

2011-06-01T00:00:00Z

Many parameters predict for outcome after unrelated donor (URD) allogeneic hematopoietic stem cell transplantation (alloSCT). High-resolution HLA-matching significantly impacts outcome and also the European Group of Blood and Marrow Transplantation (EBMT) risk score, based on patient age, disease stage, donor type, time from diagnosis to SCT and gender combination, may predict for non-relapse mortality and overall survival (OS). We evaluated the individual and combined effects of allele-matching and the EBMT risk score in 327 patients with poor-risk acute leukemia or myelodysplasia, who received a T-cell depleted URD alloSCT. Matching for HLA-A, -B, -C and -DRB1 alleles (8/8 match) was associated with a 5-year OS of 40% compared with 30% for mismatched (≤7/8) pairs (P=0.02). Patients with EBMT risk scores of 1-2, 3, 4 and 5-7 had 5-year OS estimates of 53, 43, 30 and 20%, respectively (P<0.001). The favorable prognostic impact of an 8/8 donor was most pronounced if the EBMT risk score was low (1-2). Five-year OS was 74±8% vs 39±11% for fully matched patients with a low-risk EBMT score as compared with EBMT low-risk patients with ≤7/8 donors. These data underscore the importance of incorporating both the EBMT risk score and the degree of high-resolution HLA-matching in the risk assessment prior to URD alloSCT.Leukemia advance online publication, 24 May 2011; doi:10.1038/leu.2011.123.

ATP-dependent and independent functions of Rad54 in genome maintenance (Article)

2011-03-07T00:00:00Z

Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54's ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54's ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non-DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus.

In vitro reconstitution of the functional interplay between MCAK and EB3 at microtubule plus ends (Article)

2010-10-12T00:00:00Z

The kinesin-13 family member mitotic centromere-associated kinesin (MCAK) is a potent microtubule depolymerase [1-4]. Paradoxically, in cells it accumulates at the growing, rather than the shortening, microtubule plus ends. This plus-end tracking behavior requires the interaction between MCAK and members of the end-binding protein (EB) family [5-8], but the effect of EBs on the microtubule-destabilizing activity of MCAK and the functional significance of MCAK accumulation at the growing microtubule tips have so far remained elusive. Here, we dissect the functional interplay between MCAK and EB3 by reconstituting EB3-dependent MCAK activity on dynamic microtubules in vitro. Whereas MCAK alone efficiently blocks microtubule assembly, the addition of EB3 restores robust microtubule growth, an effect that is not dependent on the binding of MCAK to EB3. At the same time, EB3 targets MCAK to growing microtubule ends by increasing its association rate with microtubule tips, a process that requires direct interaction between the two proteins. This EB3-dependent microtubule plus-end accumulation does not affect the velocity of microtubule growth or shortening but enhances the capacity of MCAK to induce catastrophes. The combination of MCAK and EB3 thus promotes rapid switching between microtubule growth and shortening, which can be important for remodeling of the microtubule cytoskeleton.

Automated analysis of time-lapse fluorescence microscopy images: From live cell images to intracellular foci (Article)

2010-08-11T00:00:00Z

Motivation: Complete, accurate and reproducible analysis of intracellular foci from fluorescence microscopy image sequences of live cells requires full automation of all processing steps involved: cell segmentation and tracking followed by foci segmentation and pattern analysis. Integrated systems for this purpose are lacking. Results: Extending our previous work in cell segmentation and tracking, we developed a new system for performing fully automated analysis of fluorescent foci in single cells. The system was validated by applying it to two common tasks: intracellular foci counting (in DNA damage repair experiments) and cell-phase identification based on foci pattern analysis (in DNA replication experiments). Experimental results show that the system performs comparably to expert human observers. Thus, it may replace tedious manual analyses for the considered tasks, and enables high-content screening.

Neuron tracing in perspective (Article)

2010-07-01T00:00:00Z

The study of the structure and function of neuronal cells and networks is of crucial importance in the endeavor to understand how the brain works. A key component in this process is the extraction of neuronal morphology from microscopic imaging data. In the past four decades, many computational methods and tools have been developed for digital reconstruction of neurons from images, with limited success. As witnessed by the growing body of literature on the subject, as well as the organization of challenging competitions in the field, the quest for a robust and fully automated system of more general applicability still continues. The aim of this work, is to contribute by surveying recent developments in the field for anyone interested in taking up the challenge. Relevant aspects discussed in the article include proposed image segmentation methods, quantitative measures of neuronal morphology, currently available software tools for various related purposes, and morphology databases.

History-dependent catastrophes regulate axonal microtubule behavior (Article)

2010-06-01T00:00:00Z

In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe [1]. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules [2]. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites [3, 4]. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP [5], regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data [6]. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.

Tracking in cell and developmental biology (Article)

2009-10-01T00:00:00Z

The past decade has seen an unprecedented data explosion in biology. It has become evident that in order to take full advantage of the potential wealth of information hidden in the data produced by even a single experiment, visual inspection and manual analysis are no longer adequate. To ensure efficiency, consistency, and completeness in data processing and analysis, computational tools are essential. Of particular importance to many modern live-cell imaging experiments is the ability to automatically track and analyze the motion of objects in time-lapse microscopy images. This article surveys the recent literature in this area. Covering all scales of microscopic observation, from cells, down to molecules, and up to entire organisms, it discusses the latest trends and successes in the development and application of computerized tracking methods in cell and developmental biology.

Intracranial internal carotid artery calcifications: Association with vascular risk factors and ischemic cerebrovascular disease (Article)

2009-01-01T00:00:00Z

BACKGROUND AND PURPOSE: Atherosclerotic calcifications are present not only in the extracranial carotid bifurcation but also in the intracranial part of the internal carotid artery. We assessed the association between intracranial internal carotid artery calcifications and cardiovascular risk factors in patients with ischemic cerebrovascular disease and the association between calcifications and the presence of this disease. MATERIALS AND METHODS: Patients undergoing multidetector CT (MDCT) angiography of the carotid arteries for assessment of stenosis degree were included in the study. A semiautomatic custom-made system to quantify calcifications was developed. The associations between the volume of calcifications and cardiovascular risk factors and the type of ischemic cerebrovascular symptoms were assessed with logistic regression. RESULTS: MDCT angiography was performed in 406 patients (age, 62 ± 14 years; 242 men). Men had a significantly higher calcification volume (66 mm3) than women (33 mm3). Calcification volume was positively associated with age in both men and women. Smoking, hypercholesterolemia, and a history of cardiac disease were independently related to the presence of calcifications. A history of cardiac disease and ischemic cerebrovascular disease were independently related to the volume of calcifications. No association was found between calcifications and the presence or type of ischemic cerebrovascular disease in the vascular territory of the intracranial internal carotid artery. CONCLUSIONS: Calcifications were associated with higher age and male gender. The presence and volume of calcifications were independently associated with cardiovascular risk factors. Calcifications were not related to the presence or type of ischemic cerebrovascular disease.

Multiple object tracking in molecular bioimaging by Rao-Blackwellized marginal particle filtering (Article)

2008-12-01T00:00:00Z

Time-lapse fluorescence microscopy imaging has rapidly evolved in the past decade and has opened new avenues for studying intracellular processes in vivo. Such studies generate vast amounts of noisy image data that cannot be analyzed efficiently and reliably by means of manual processing. Many popular tracking techniques exist but often fail to yield satisfactory results in the case of high object densities, high noise levels, and complex motion patterns. Probabilistic tracking algorithms, based on Bayesian estimation, have recently been shown to offer several improvements over classical approaches, by better integration of spatial and temporal information, and the possibility to more effectively incorporate prior knowledge about object dynamics and image formation. In this paper, we extend our previous work in this area and propose an improved, fully automated particle filtering algorithm for the tracking of many subresolution objects in fluorescence microscopy image sequences. It involves a new track management procedure and allows the use of multiple dynamics models. The accuracy and reliability of the algorithm are further improved by applying marginalization concepts. Experiments on synthetic as well as real image data from three different biological applications clearly demonstrate the superiority of the algorithm compared to previous particle filtering solutions.

A new detection scheme for multiple object tracking in fluorescence microscopy by joint probabilistic data association filtering (In Proceedings)

2008-09-10T00:00:00Z

Tracking of multiple objects in biological image data is a challenging problem due largely to poor imaging conditions and complicated motion scenarios. Existing tracking algorithms for this purpose often do not provide sufficient robustness and/or are computationally expensive. In this paper we propose a new object detection scheme, based on importance sampling from image intensity distributions, and show how it can be easily incorporated into a probabilistic tracking framework based on Kalman or particle filtering. Experiments on synthetic as well as real fluorescence microscopy image data from different biological studies show that the resulting tracking algorithm yields smaller localization errors at much lower execution times compared to other available methods.

Advanced level-set based multiple-cell segmentation and tracking in time-lapse fluorescence microscopy images (In Proceedings)

2008-09-10T00:00:00Z

Segmentation and tracking of cells in fluorescence microscopy image sequences is an important task in many biological studies into cell migration as well as intracellular dynamics. The growing size and complexity of biological image data sets precludes manual analysis, and calls for increasingly advanced automatic algorithms that are generic enough to be easily tunable to different applications, yet robust enough to deal with different cell types and strongly varying imaging conditions. Active-contour based algorithms have proven to be very suitable for this purpose but still suffer from several short-comings that limit their segmentation accuracy and tracking robustness. In addition, these algorithms are generally rather computationally expensive. In this paper, we present an advanced level-set based multiple-cell segmentation and tracking algorithm, which implements seven modifications compared to earlier algorithms that considerably improve its performance. Preliminary experiments on three different time-lapse fluorescence microscopy images demonstrate the potential of the new algorithm.

Assessment of atherosclerotic carotid plaque volume with multidetector computed tomography angiography (Article)

2008-03-31T00:00:00Z

Purpose: The amount of atherosclerotic plaque and its components (calcifications, fibrous tissue, and lipid core) could be better predictors of acute events than the now currently used degree of stenosis. Therefore, we evaluated a dedicated software tool for volume measurements of atherosclerotic carotid plaque and its components in multidetector computed tomography angiography (MDCTA) images. Materials and Methods: Data acquisition was approved by the Institutional Review Board and all patients gave written informed consent. MDCTA images of 56 carotid arteries were analyzed by three observers. Plaque volumes were assessed by manual drawing of the outer vessel contour. The luminal boundary was determined based on a Hounsfield-Unit (HU) threshold. The contribution of different components was measured by the number of voxels within defined ranges of HU-values (calcification >130 HU, fibrous tissue 60-130 HU, lipid core <60 HU). Interobserver variability (IOV) was assessed. Results: Plaque volume was 1,259 ± 621 mm3. The calcified, fibrous and lipid volumes were 238 ± 252 mm3, 647± 277 mm3 and 376± 283 mm3, respectively. IOV was moderate with interclass correlation coefficients (ICC) ranging from 0.76 to 0.99 and coefficients of variation (COV) ranging from 3% to 47%. Conclusion: Atherosclerotic carotid plaque volume and plaque component volumes can be assessed with MDCTA with a reasonable observer variability.

Evaluation of an improved technique for lumen path definition and lumen segmentation of atherosclerotic vessels in CT angiography (Article)

2007-07-01T00:00:00Z

Vessel image analysis is crucial when considering therapeutical options for (cardio-) vascular diseases. Our method, VAMPIRE (Vascular Analysis using Multiscale Paths Inferred from Ridges and Edges), involves two parts: a user defines a start- and endpoint upon which a lumen path is automatically defined, and which is used for initialization; the automatic segmentation of the vessel lumen on computed tomographic angiography (CTA) images. Both parts are based on the detection of vessel-like structures by analyzing intensity, edge, and ridge information. A multi-observer evaluation study was performed to compare VAMPIRE with a conventional method on the CTA data of 15 patients with carotid artery stenosis. In addition to the start- and endpoint, the two radiologists required on average 2.5 (SD: 1.9) additional points to define a lumen path when using the conventional method, and 0.1 (SD: 0.3) when using VAMPIRE. The segmentation results were quantitatively evaluated using Similarity Indices, which were slightly lower between VAMPIRE and the two radiologists (respectively 0.90 and 0.88) compared with the Similarity Index between the radiologists (0.92). The evaluation shows that the improved definition of a lumen path requires minimal user interaction, and that using this path as initialization leads to good automatic lumen segmentation results.