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64
Tracking in molecular bioimaging
- IEEE Signal Processing Mag
, 2006
"... Automated tracking and analysis of moving objects in image sequences has been and continues to be one of the major themes in digital image analysis research. This is not surprising in view of its many applications in video surveillance, multimedia services, automated vehicle guidance and driver assi ..."
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Automated tracking and analysis of moving objects in image sequences has been and continues to be one of the major themes in digital image analysis research. This is not surprising in view of its many applications in video surveillance, multimedia services, automated vehicle guidance and driver assistance, remote sensing and meteorology, and medical imaging. It is also a recurring theme in molecular biology. By their very nature, biomolecular systems are dynamic, and it is one of the major challenges of biomedical research and pharmaceutical industries in the postgenomic era to unravel the spatial and temporal relationships of these complex systems and to devise strategies to manipulate them. Results in this area can be expected to have profound social and economic impact in the near future, as they can be harnessed to improve human health and well-being [1]. Studies into biomolecular dynamics generate ever increasing amounts of image data. To be able to handle these data and to fully exploit them for describing biological processes on a quantitative level and building accurate mathematical models of dynamic structures, computerized motion analysis is rapidly becoming a requisite [2]. Over the past decades, a number of image analysis techniques have been developed in support of such studies, the details of which were often buried in the small print of the methods sections of papers published in the biology and biophysics literature. The majority of these techniques were based on rather rudimentary
Intranuclear binding kinetics and mobility of single native U1 snRNP particles in living cells
- Mol. Biol. Cell
, 2006
"... Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single ..."
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Cited by 9 (2 self)
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Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay behavior on time scales ranging from milliseconds to seconds, reflecting the involvement of U1 snRNPs in numerous distinct interactions. The average dwell times for U1 snRNPs bound at sites within the nucleoplasm did not differ significantly from those in speckles, indicating that similar processes occur in both compartments. Mobile U1 snRNPs moved with diffusion constants in the range from 0.5 to 8 m2/s. These values were consistent with uncomplexed U1 snRNPs diffusing at a viscosity of 5 cPoise and U1 snRNPs moving in a largely restricted manner, and U1 snRNPs contained in large supramolecular assemblies such as spliceosomes or supraspliceosomes.
Segmentation of whole cells and cell nuclei from 3-D optical microscope images using dynamic programming
- IEEE Trans. Med. Imag
, 2008
"... Abstract—Communications between cells in large part drive tissue development and function, as well as disease-related pro-cesses such as tumorigenesis. Understanding the mechanistic bases of these processes necessitates quantifying specific molecules in adjacent cells or cell nuclei of intact tissue ..."
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Abstract—Communications between cells in large part drive tissue development and function, as well as disease-related pro-cesses such as tumorigenesis. Understanding the mechanistic bases of these processes necessitates quantifying specific molecules in adjacent cells or cell nuclei of intact tissue. However, a major restriction on such analyses is the lack of an efficient method that correctly segments each object (cell or nucleus) from 3-D images of an intact tissue specimen. We report a highly reliable and accurate semi-automatic algorithmic method for segmenting fluorescence-labeled cells or nuclei from 3-D tissue images. Seg-mentation begins with semi-automatic, 2-D object delineation in a user-selected plane, using dynamic programming (DP) to locate the border with an accumulated intensity per unit length greater that any other possible border around the same object. Then the two surfaces of the object in planes above and below the selected plane are found using an algorithm that combines DP and combinatorial searching. Following segmentation, any perceived
RNA interference and single particle tracking analysis of hepatitis C virus endocytosis. PLoS Pathog
- 2009
"... Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a s ..."
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Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with
A Dynamical Model Reveals Gene Co-Localizations in Nucleus
, 2010
"... Co-localization of networks of genes in the nucleus is thought to play an important role in determining gene expression patterns. Based upon experimental data, we built a dynamical model to test whether pure diffusion could account for the observed co-localization of genes within a defined subnuclea ..."
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Co-localization of networks of genes in the nucleus is thought to play an important role in determining gene expression patterns. Based upon experimental data, we built a dynamical model to test whether pure diffusion could account for the observed co-localization of genes within a defined subnuclear region. A simple standard Brownian motion model in two and three dimensions shows that preferential co-localization is possible for co-regulated genes without any direct interaction, and suggests the occurrence may be due to a limitation in the number of available transcription factors. Experimental data of chromatin movements demonstrates that fractional rather than standard Brownian motion is more appropriate to model gene mobilizations, and we tested our dynamical model against recent static experimental data, using a sub-diffusion process by which the genes tend to colocalize more easily. Moreover, in order to compare our model with recently obtained experimental data, we studied the association level between genes and factors, and presented data supporting the validation of this dynamic model. As further applications of our model, we applied it to test against more biological observations. We found that increasing transcription factor number, rather than factory number and nucleus size, might be the reason for decreasing gene co-localization. In the scenario of frequency- or amplitude-modulation of transcription factors, our model predicted that frequency-modulation may increase the co-localization between its targeted
Microtubule dynamics analysis using kymographs and variable-rate particle filters
- IEEE Trans. Image Process
, 2010
"... Abstract—Studying intracellular dynamics is of fundamental importance for understanding healthy life at the molecular level and for developing drugs to target disease processes. One of the key technologies to enable this research is the automated tracking and motion analysis of these objects in micr ..."
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Abstract—Studying intracellular dynamics is of fundamental importance for understanding healthy life at the molecular level and for developing drugs to target disease processes. One of the key technologies to enable this research is the automated tracking and motion analysis of these objects in microscopy image sequences. To make better use of the spatiotemporal information than common frame-by-frame tracking methods, two alterna-tive approaches have recently been proposed, based upon either Bayesian estimation or space-time segmentation. In this paper, we propose to combine the power of both approaches, and develop a new probabilistic method to segment the traces of the moving objects in kymograph representations of the image data. It is based on variable-rate particle filtering and uses multiscale trend analysis of the extracted traces to estimate the relevant kinematic parameters. Experiments on realistic synthetically generated images as well as on real biological image data demonstrate the improved potential of the new method for the analysis of micro-tubule dynamics in vitro. Index Terms—Bayesian estimation, biological microscopy, microtubule dynamics, motion analysis, multiscale trend analysis, variable-rate particle filters. I.
Characterization of Eag1 Channel Lateral Mobility in Rat Hippocampal Cultures by Single-Particle-Tracking with Quantum Dots
"... Voltage-gated ion channels are main players involved in fast synaptic events. However, only slow intracellular mechanisms have so far been described for controlling their localization as real-time visualization of endogenous voltage-gated channels at high temporal and spatial resolution has not been ..."
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Voltage-gated ion channels are main players involved in fast synaptic events. However, only slow intracellular mechanisms have so far been described for controlling their localization as real-time visualization of endogenous voltage-gated channels at high temporal and spatial resolution has not been achieved yet. Using a specific extracellular antibody and quantum dots we reveal and characterize lateral mobility as a faster mechanism to dynamically control the number of endogenous ethera-go-go (Eag)1 ion channels inside synapses. We visualize Eag1 entering and leaving synapses by lateral diffusion in the plasma membrane of rat hippocampal neurons. Mathematical analysis of their trajectories revealed how the motion of Eag1 gets restricted when the channels diffuse into the synapse, suggesting molecular interactions between Eag1 and synaptic components. In contrast, Eag1 channels switch to Brownian movement when they exit synapses and diffuse into extrasynaptic membranes. Furthermore, we demonstrate that the mobility of Eag1 channels is specifically regulated inside synapses by actin filaments, microtubules and electrical activity. In summary, using single-particle-tracking techniques with quantum dots nanocrystals, our study shows for the first time the lateral diffusion of an endogenous voltage-gated ion channel in neurons. The location-dependent constraints imposed by cytoskeletal elements together with the regulatory role
Tracking of Virus Particles in Time-Lapse Fluorescence Microscopy Image Sequences
"... Abstract. Modern developments in time-lapse microscopy enable the observation of a variety of processes exhibited by viruses. The dynamic nature of these processes requires the tracking of viruses over time to explore the spatio-temporal relationships. In this work, we developed deterministic and pr ..."
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Abstract. Modern developments in time-lapse microscopy enable the observation of a variety of processes exhibited by viruses. The dynamic nature of these processes requires the tracking of viruses over time to explore the spatio-temporal relationships. In this work, we developed deterministic and probabilistic approaches for multiple virus tracking. A quantitative comparison based on synthetic image sequences was carried out to evaluate the performance of the different algorithms. We have also applied the algorithms to real microscopy images of HIV-1 particles and have compared the tracking results with ground truth obtained from manual tracking. It turns out that the probabilistic approach outperforms the deterministic schemes. 1
Synchronized retrovirus fusion in cells expressing alternative receptor isoforms releases the viral core into distinct sub-cellular compartments. PLoS Pathog
"... Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian ..."
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Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude
