Significantly improved precision of cell migration analysis in time-lapse video microscopy through use of a fully automated tracking system

<p>Abstract</p> <p>Background</p> <p>Cell motility is a critical parameter in many physiological as well as pathophysiological processes. In time-lapse video microscopy, manual cell tracking remains the most common method of analyzing migratory behavior of cell populations. In addition to being labor-intensive, this method is susceptible to user-dependent errors regarding the selection of "representative" subsets of cells and manual determination of precise cell positions.</p> <p>Results</p> <p>We have quantitatively analyzed these error sources, demonstrating that manual cell tracking of pancreatic cancer cells lead to mis-calculation of migration rates of up to 410%. In order to provide for objective measurements of cell migration rates, we have employed multi-target tracking technologies commonly used in radar applications to develop fully automated cell identification and tracking system suitable for high throughput screening of video sequences of unstained living cells.</p> <p>Conclusion</p> <p>We demonstrate that our automatic multi target tracking system identifies cell objects, follows individual cells and computes migration rates with high precision, clearly outperforming manual procedures.</p

Particle detection and tracking in fluorescence time-lapse imaging: a contrario approach

This paper proposes a probabilistic approach for the detection and the tracking of particles in fluorescent time-lapse imaging. In the presence of a very noised and poor-quality data, particles and trajectories can be characterized by an a contrario model, that estimates the probability of observing the structures of interest in random data. This approach, first introduced in the modeling of human visual perception and then successfully applied in many image processing tasks, leads to algorithms that neither require a previous learning stage, nor a tedious parameter tuning and are very robust to noise. Comparative evaluations against a well-established baseline show that the proposed approach outperforms the state of the art.Comment: Published in Journal of Machine Vision and Application

Automated Deep Lineage Tree Analysis Using a Bayesian Single Cell Tracking Approach

Single-cell methods are beginning to reveal the intrinsic heterogeneity in cell populations, arising from the interplay of deterministic and stochastic processes. However, it remains challenging to quantify single-cell behaviour from time-lapse microscopy data, owing to the difficulty of extracting reliable cell trajectories and lineage information over long time-scales and across several generations. Therefore, we developed a hybrid deep learning and Bayesian cell tracking approach to reconstruct lineage trees from live-cell microscopy data. We implemented a residual U-Net model coupled with a classification CNN to allow accurate instance segmentation of the cell nuclei. To track the cells over time and through cell divisions, we developed a Bayesian cell tracking methodology that uses input features from the images to enable the retrieval of multi-generational lineage information from a corpus of thousands of hours of live-cell imaging data. Using our approach, we extracted 20,000 + fully annotated single-cell trajectories from over 3,500 h of video footage, organised into multi-generational lineage trees spanning up to eight generations and fourth cousin distances. Benchmarking tests, including lineage tree reconstruction assessments, demonstrate that our approach yields high-fidelity results with our data, with minimal requirement for manual curation. To demonstrate the robustness of our minimally supervised cell tracking methodology, we retrieve cell cycle durations and their extended inter- and intra-generational family relationships in 5,000 + fully annotated cell lineages. We observe vanishing cycle duration correlations across ancestral relatives, yet reveal correlated cyclings between cells sharing the same generation in extended lineages. These findings expand the depth and breadth of investigated cell lineage relationships in approximately two orders of magnitude more data than in previous studies of cell cycle heritability, which were reliant on semi-manual lineage data analysis

Local cellular neighbourhood controls proliferation in cell competition

Cell competition is a quality control mechanism through which tissues eliminate unfit cells. Cell competition can result from short-range biochemical inductions or long-range mechanical cues. However, little is known about how cell-scale interactions give rise to population shifts in tissues, due to the lack of experimental and computational tools to efficiently characterise interactions at the single-cell level. Here, we address these challenges by combining long-term automated microscopy with deep learning image analysis to decipher how single-cell behaviour determines tissue make-up during competition. Using our high-throughput analysis pipeline, we show that competitive interactions between MDCK wild-type cells and cells depleted of the polarity protein scribble are governed by differential sensitivity to local density and the cell-type of each cell's neighbours. We find that local density has a dramatic effect on the rate of division and apoptosis under competitive conditions. Strikingly, our analysis reveals that proliferation of the winner cells is upregulated in neighbourhoods mostly populated by loser cells. These data suggest that tissue-scale population shifts are strongly affected by cellular-scale tissue organisation. We present a quantitative mathematical model that demonstrates the effect of neighbour cell-type dependence of apoptosis and division in determining the fitness of competing cell lines

Cyclist Detection, Tracking, and Trajectory Analysis in Urban Traffic Video Data

The major objective of this thesis work is examining computer vision and machine learning detection methods, tracking algorithms and trajectory analysis for cyclists in traffic video data and developing an efficient system for cyclist counting. Due to the growing number of cyclist accidents on urban roads, methods for collecting information on cyclists are of significant importance to the Department of Transportation. The collected information provides insights into solving critical problems related to transportation planning, implementing safety countermeasures, and managing traffic flow efficiently. Intelligent Transportation System (ITS) employs automated tools to collect traffic information from traffic video data. In comparison to other road users, such as cars and pedestrians, the automated cyclist data collection is relatively a new research area. In this work, a vision-based method for gathering cyclist count data at intersections and road segments is developed. First, we develop methodology for an efficient detection and tracking of cyclists. The combination of classification features along with motion based properties are evaluated to detect cyclists in the test video data. A Convolutional Neural Network (CNN) based detector called You Only Look Once (YOLO) is implemented to increase the detection accuracy. In the next step, the detection results are fed into a tracker which is implemented based on the Kernelized Correlation Filters (KCF) which in cooperation with the bipartite graph matching algorithm allows to track multiple cyclists, concurrently. Then, a trajectory rebuilding method and a trajectory comparison model are applied to refine the accuracy of tracking and counting. The trajectory comparison is performed based on semantic similarity approach. The proposed counting method is the first cyclist counting method that has the ability to count cyclists under different movement patterns. The trajectory data obtained can be further utilized for cyclist behavioral modeling and safety analysis

Motion patterns of subviral particles: Digital tracking, image data processing and analysis

At the Institute of Virology, Philipps-University, Marburg, Germany, currently research on the understanding of the transport mechanisms of Ebola- and Marburgvirus nucleocapsids is carried out. This research demands a profound knowledge about the various motion characteristics of the nucleocapids. The analysis of large amounts of samples by conventional manual evaluation is a laborious task and does not always lead to reproducible and comparable results. In a cooperation between the Institute of Virology, Marburg, and the Institute for Biomedical Engineering, University of Applied Sciences, Giessen, Germany, algorithms are developed and programmed that enable an automatic tracking of subviral particles in fluorescence microscopic image sequences. The algorithms form an interface between the biologic and the algorithmic domain. Furthermore, methods to automatically parameterize and classify subviral particle motions are created. Geometric and mathematical approaches, like curvature-, fractal dimension- and mean squared displacement-determination are applied. Statistical methods are used to compare the measured subviral particle motion parameters between different biological samples. In this thesis, the biological, mathematical and algorithmic basics are described and the state of the art methods of other research groups are presented and compared. The algorithms to track, parameterize, classify and statistically analyze subviral particle tracks are presented in the Methods section. All methods are evaluated with simulated data and/or compared to data validated by a virologist. The methods are applied to a set of real fluorescence microscopic image sequences of Marburgvirus infected live-cells. The Results chapter shows that subviral particle motion can be successfully analyzed using the presented tracking and analysis methods. Furthermore, differences between the subviral particle motions in the analyzed groups could be detected. However, further optimization with manually evaluated data can improve the results. The methods developed in this project enhance the knowledge about nucleocapsid transport and may be valuable for the development of effective antiviral agents to cure Ebola- and Marburgvirus diseases. The thesis concludes with a chapter Discussion and Conclusions

Radar-based Application of Pedestrian and Cyclist Micro-Doppler Signatures for Automotive Safety Systems

Die sensorbasierte Erfassung des Nahfeldes im Kontext des hochautomatisierten Fahrens erfährt einen spürbaren Trend bei der Integration von Radarsensorik. Fortschritte in der Mikroelektronik erlauben den Einsatz von hochauflösenden Radarsensoren, die durch effiziente Verfahren sowohl im Winkel als auch in der Entfernung und im Doppler die Messgenauigkeit kontinuierlich ansteigen lassen. Dadurch ergeben sich neuartige Möglichkeiten bei der Bestimmung der geometrischen und kinematischen Beschaffenheit ausgedehnter Ziele im Fahrzeugumfeld, die zur gezielten Entwicklung von automotiven Sicherheitssystemen herangezogen werden können. Im Rahmen dieser Arbeit werden ungeschützte Verkehrsteilnehmer wie Fußgänger und Radfahrer mittels eines hochauflösenden Automotive-Radars analysiert. Dabei steht die Erscheinung des Mikro-Doppler-Effekts, hervorgerufen durch das hohe Maß an kinematischen Freiheitsgraden der Objekte, im Vordergrund der Betrachtung. Die durch den Mikro-Doppler-Effekt entstehenden charakteristischen Radar-Signaturen erlauben eine detailliertere Perzeption der Objekte und können in direkten Zusammenhang zu ihren aktuellen Bewegungszuständen gesetzt werden. Es werden neuartige Methoden vorgestellt, die die geometrischen und kinematischen Ausdehnungen der Objekte berücksichtigen und echtzeitfähige Ansätze zur Klassifikation und Verhaltensindikation realisieren. Wird ein ausgedehntes Ziel (z.B. Radfahrer) von einem Radarsensor detektiert, können aus dessen Mikro-Doppler-Signatur wesentliche Eigenschaften bezüglich seines Bewegungszustandes innerhalb eines Messzyklus erfasst werden. Die Geschwindigkeitsverteilungen der sich drehenden Räder erlauben eine adaptive Eingrenzung der Tretbewegung, deren Verhalten essentielle Merkmale im Hinblick auf eine vorausschauende Unfallprädiktion aufweist. Ferner unterliegen ausgedehnte Radarziele einer Orientierungsabhängigkeit, die deren geometrischen und kinematischen Profile direkt beeinflusst. Dies kann sich sowohl negativ auf die Klassifikations-Performance als auch auf die Verwertbarkeit von Parametern auswirken, die eine Absichtsbekundung des Radarziels konstituieren. Am Beispiel des Radfahrers wird hierzu ein Verfahren vorgestellt, das die orientierungsabhängigen Parameter in Entfernung und Doppler normalisiert und die gemessenen Mehrdeutigkeiten kompensiert. Ferner wird in dieser Arbeit eine Methodik vorgestellt, die auf Grundlage des Mikro- Doppler-Profils eines Fußgängers dessen Beinbewegungen über die Zeit schätzt (Tracking) und wertvolle Objektinformationen hinsichtlich seines Bewegungsverhaltens offenbart. Dazu wird ein Bewegungsmodell entwickelt, das die nichtlineare Fortbewegung des Beins approximiert und dessen hohes Maß an biomechanischer Variabilität abbildet. Durch die Einbeziehung einer wahrscheinlichkeitsbasierten Datenassoziation werden die Radar-Detektionen ihren jeweils hervorrufenden Quellen (linkes und rechtes Bein) zugeordnet und eine Trennung der Gliedmaßen realisiert. Im Gegensatz zu bisherigen Tracking-Verfahren weist die vorgestellte Methodik eine Steigerung in der Genauigkeit der Objektinformationen auf und stellt damit einen entscheidenden Vorteil für zukünftige Fahrerassistenzsysteme dar, um deutlich schneller auf kritische Verkehrssituationen reagieren zu können.:1 Introduction 1 1.1 Automotive environmental perception 2 1.2 Contributions of this work 4 1.3 Thesis overview 6 2 Automotive radar 9 2.1 Physical fundamentals 9 2.1.1 Radar cross section 9 2.1.2 Radar equation 10 2.1.3 Micro-Doppler effect 11 2.2 Radar measurement model 15 2.2.1 FMCW radar 15 2.2.2 Chirp sequence modulation 17 2.2.3 Direction-of-arrival estimation 22 2.3 Signal processing 25 2.3.1 Target properties 26 2.3.2 Target extraction 28 Power detection 28 Clustering 30 2.3.3 Real radar data example 31 2.4 Conclusion 33 3 Micro-Doppler applications of a cyclist 35 3.1 Physical fundamentals 35 3.1.1 Micro-Doppler signatures of a cyclist 35 3.1.2 Orientation dependence 36 3.2 Cyclist feature extraction 38 3.2.1 Adaptive pedaling extraction 38 Ellipticity constraints 38 Ellipse fitting algorithm 39 3.2.2 Experimental results 42 3.3 Normalization of the orientation dependence 44 3.3.1 Geometric correction 44 3.3.2 Kinematic correction 45 3.3.3 Experimental results 45 3.4 Conclusion 47 3.5 Discussion and outlook 47 4 Micro-Doppler applications of a pedestrian 49 4.1 Pedestrian detection 49 4.1.1 Human kinematics 49 4.1.2 Micro-Doppler signatures of a pedestrian 51 4.1.3 Experimental results 52 Radially moving pedestrian 52 Crossing pedestrian 54 4.2 Pedestrian feature extraction 57 4.2.1 Frequency-based limb separation 58 4.2.2 Extraction of body parts 60 4.2.3 Experimental results 62 4.3 Pedestrian tracking 64 4.3.1 Probabilistic state estimation 65 4.3.2 Gaussian filters 67 4.3.3 The Kalman filter 67 4.3.4 The extended Kalman filter 69 4.3.5 Multiple-object tracking 71 4.3.6 Data association 74 4.3.7 Joint probabilistic data association 80 4.4 Kinematic-based pedestrian tracking 84 4.4.1 Kinematic modeling 84 4.4.2 Tracking motion model 87 4.4.3 4-D radar point cloud 91 4.4.4 Tracking implementation 92 4.4.5 Experimental results 96 Longitudinal trajectory 96 Crossing trajectory with sudden turn 98 4.5 Conclusion 102 4.6 Discussion and outlook 103 5 Summary and outlook 105 5.1 Developed algorithms 105 5.1.1 Adaptive pedaling extraction 105 5.1.2 Normalization of the orientation dependence 105 5.1.3 Model-based pedestrian tracking 106 5.2 Outlook 106 Bibliography 109 List of Acronyms 119 List of Figures 124 List of Tables 125 Appendix 127 A Derivation of the rotation matrix 2.26 127 B Derivation of the mixed radar signal 2.52 129 C Calculation of the marginal association probabilities 4.51 131 Curriculum Vitae 135Sensor-based detection of the near field in the context of highly automated driving is experiencing a noticeable trend in the integration of radar sensor technology. Advances in microelectronics allow the use of high-resolution radar sensors that continuously increase measurement accuracy through efficient processes in angle as well as distance and Doppler. This opens up novel possibilities in determining the geometric and kinematic nature of extended targets in the vehicle environment, which can be used for the specific development of automotive safety systems. In this work, vulnerable road users such as pedestrians and cyclists are analyzed using a high-resolution automotive radar. The focus is on the appearance of the micro-Doppler effect, caused by the objects’ high kinematic degree of freedom. The characteristic radar signatures produced by the micro-Doppler effect allow a clearer perception of the objects and can be directly related to their current state of motion. Novel methods are presented that consider the geometric and kinematic extents of the objects and realize real-time approaches to classification and behavioral indication. When a radar sensor detects an extended target (e.g., bicyclist), its motion state’s fundamental properties can be captured from its micro-Doppler signature within a measurement cycle. The spinning wheels’ velocity distributions allow an adaptive containment of the pedaling motion, whose behavior exhibits essential characteristics concerning predictive accident prediction. Furthermore, extended radar targets are subject to orientation dependence, directly affecting their geometric and kinematic profiles. This can negatively affect both the classification performance and the usability of parameters constituting the radar target’s intention statement. For this purpose, using the cyclist as an example, a method is presented that normalizes the orientation-dependent parameters in range and Doppler and compensates for the measured ambiguities. Furthermore, this paper presents a methodology that estimates a pedestrian’s leg motion over time (tracking) based on the pedestrian’s micro-Doppler profile and reveals valuable object information regarding his motion behavior. To this end, a motion model is developed that approximates the leg’s nonlinear locomotion and represents its high degree of biomechanical variability. By incorporating likelihood-based data association, radar detections are assigned to their respective evoking sources (left and right leg), and limb separation is realized. In contrast to previous tracking methods, the presented methodology shows an increase in the object information’s accuracy. It thus represents a decisive advantage for future driver assistance systems in order to be able to react significantly faster to critical traffic situations.:1 Introduction 1 1.1 Automotive environmental perception 2 1.2 Contributions of this work 4 1.3 Thesis overview 6 2 Automotive radar 9 2.1 Physical fundamentals 9 2.1.1 Radar cross section 9 2.1.2 Radar equation 10 2.1.3 Micro-Doppler effect 11 2.2 Radar measurement model 15 2.2.1 FMCW radar 15 2.2.2 Chirp sequence modulation 17 2.2.3 Direction-of-arrival estimation 22 2.3 Signal processing 25 2.3.1 Target properties 26 2.3.2 Target extraction 28 Power detection 28 Clustering 30 2.3.3 Real radar data example 31 2.4 Conclusion 33 3 Micro-Doppler applications of a cyclist 35 3.1 Physical fundamentals 35 3.1.1 Micro-Doppler signatures of a cyclist 35 3.1.2 Orientation dependence 36 3.2 Cyclist feature extraction 38 3.2.1 Adaptive pedaling extraction 38 Ellipticity constraints 38 Ellipse fitting algorithm 39 3.2.2 Experimental results 42 3.3 Normalization of the orientation dependence 44 3.3.1 Geometric correction 44 3.3.2 Kinematic correction 45 3.3.3 Experimental results 45 3.4 Conclusion 47 3.5 Discussion and outlook 47 4 Micro-Doppler applications of a pedestrian 49 4.1 Pedestrian detection 49 4.1.1 Human kinematics 49 4.1.2 Micro-Doppler signatures of a pedestrian 51 4.1.3 Experimental results 52 Radially moving pedestrian 52 Crossing pedestrian 54 4.2 Pedestrian feature extraction 57 4.2.1 Frequency-based limb separation 58 4.2.2 Extraction of body parts 60 4.2.3 Experimental results 62 4.3 Pedestrian tracking 64 4.3.1 Probabilistic state estimation 65 4.3.2 Gaussian filters 67 4.3.3 The Kalman filter 67 4.3.4 The extended Kalman filter 69 4.3.5 Multiple-object tracking 71 4.3.6 Data association 74 4.3.7 Joint probabilistic data association 80 4.4 Kinematic-based pedestrian tracking 84 4.4.1 Kinematic modeling 84 4.4.2 Tracking motion model 87 4.4.3 4-D radar point cloud 91 4.4.4 Tracking implementation 92 4.4.5 Experimental results 96 Longitudinal trajectory 96 Crossing trajectory with sudden turn 98 4.5 Conclusion 102 4.6 Discussion and outlook 103 5 Summary and outlook 105 5.1 Developed algorithms 105 5.1.1 Adaptive pedaling extraction 105 5.1.2 Normalization of the orientation dependence 105 5.1.3 Model-based pedestrian tracking 106 5.2 Outlook 106 Bibliography 109 List of Acronyms 119 List of Figures 124 List of Tables 125 Appendix 127 A Derivation of the rotation matrix 2.26 127 B Derivation of the mixed radar signal 2.52 129 C Calculation of the marginal association probabilities 4.51 131 Curriculum Vitae 13

Design, implementation and evaluation of automated surveillance systems

El reconocimiento de patrones ha conseguido un nivel de complejidad que nos permite reconocer diferente tipo de eventos, incluso peligros, y actuar en concordancia para minimizar el impacto de una situación complicada y abordarla de la mejor manera posible. Sin embargo, creemos que todavía se puede llegar a alcanzar aplicaciones más eficientes con algoritmos más precisos. Nuestra aplicación quiere probar a incluir el nuevo paradigma de la programación, las redes neuronales. Nuestra idea en principio fue explorar la alternativa que las nuevas redes neuronales convolucionales aportaban, en donde se podía ver en vídeos de ejemplos la alta tasa de detección e identificación que, por ejemplo, YOLOv2 podría mostrar. Después de comparar las características, vimos que YOLOv3 ofrecía un buen balance entre precisión y rapidez como comentaremos más adelante. Debido a la tasa de baja detecciones, haremos uso de los filtros de Kalman para ayudarnos a la hora de hacer reidentificación de personas y objetos. En este proyecto, haremos un estudio además de las alternativas de videovigilancia con las que cuentan empresas del sector y veremos que clase de productos ofrecen y, por otro lado, observaremos cuales son los trabajos de los grupos de investigadores de otras universidades que más similitudes tienen con nuestro objetivo. Dedicaremos, por lo tanto, el uso de esta red neuronal para detectar eventos como el abandono de mochilas y para mostrar la densidad de tránsito en localizaciones concretas, así como utilizaremos una metodología más tradicional, el flujo óptico, para detectar actuaciones anormales en una multitud.Automatic surveillance system is getting more and more sophisticated with the increasing calculation power that computers are reaching. The aim of this project is to take advantage of these tools and with the new classification and detection technology brought by neural networks, develop a surveillance application that can recognize certain behaviours (which are the detection of lost backpacks and suitcases, detection of abnormal crowd activity and heatmap of density occupation). To develop this program, python has been the selected programming language used, where YOLO and OpenCV form the spine of this project. After testing the code, it has been proved that due to the constrains of the detection for small objects, the project does not perform as it should for real development, but still it shows potential for the detection of lost backpacks in certain videos from the GBA dataset [1] and PETS2006 dataset [2]. The abnormal activity detection for crowds is made with a simple algorithm that seems to perform well, detecting the anomalies in all the testing dataset used, generated by the University of Minnesota [3]. Finally, the heatmap can display correctly the projection of people on the ground for five second, just as intended. The objective of this software is to be part of the core of what could be a future application with more modules that will be able to perform full automated surveillance tasks and gather useful information data, and these advances and future proposal will be explained in this memory.Máster Universitario en Ingeniería Industrial (M141