3D + t Morphological Processing: Applications to Embryogenesis Image Analysis

We propose to directly process 3D + t image sequences with mathematical morphology operators, using a new classi?cation of the 3D+t structuring elements. Several methods (?ltering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebra?sh embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebra?sh early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it bene?ts from the intrinsic redundancy of the temporal dimension, and minimizes the needs for human intervention in semi-automatic algorithms.

Spatio-temporal filtering with morphological operators for robust cell migration estimation in "in-vivo" images

The understanding of the embryogenesis in living systems requires reliable quantitative analysis of the cell migration throughout all the stages of development. This is a major challenge of the "in-toto" reconstruction based on different modalities of "in-vivo" imaging techniques -spatio-temporal resolution and image artifacts and noise. Several methods for cell tracking are available, but expensive manual interaction -time and human resources- is always required to enforce coherence. Because of this limitation it is necessary to restrict the experiments or assume an uncontrolled error rate. Is it possible to obtain automated reliable measurements of migration? can we provide a seed for biologists to complete cell lineages efficiently? We propose a filtering technique that considers trajectories as spatio-temporal connected structures that prunes out those that might introduce noise and false positives by using multi-dimensional morphological operators.

Methods for the analysis of multi-scale cell dynamics from fluorescence microscopy images

La embriogénesis es el proceso mediante el cual una célula se convierte en un ser un vivo. A lo largo de diferentes etapas de desarrollo, la población de células va proliferando a la vez que el embrión va tomando forma y se configura. Esto es posible gracias a la acción de varios procesos genéticos, bioquímicos y mecánicos que interaccionan y se regulan entre ellos formando un sistema complejo que se organiza a diferentes escalas espaciales y temporales. Este proceso ocurre de manera robusta y reproducible, pero también con cierta variabilidad que permite la diversidad de individuos de una misma especie. La aparición de la microscopía de fluorescencia, posible gracias a proteínas fluorescentes que pueden ser adheridas a las cadenas de expresión de las células, y los avances en la física óptica de los microscopios han permitido observar este proceso de embriogénesis in-vivo y generar secuencias de imágenes tridimensionales de alta resolución espacio-temporal. Estas imágenes permiten el estudio de los procesos de desarrollo embrionario con técnicas de análisis de imagen y de datos, reconstruyendo dichos procesos para crear la representación de un embrión digital. Una de las más actuales problemáticas en este campo es entender los procesos mecánicos, de manera aislada y en interacción con otros factores como la expresión genética, para que el embrión se desarrolle. Debido a la complejidad de estos procesos, estos problemas se afrontan mediante diferentes técnicas y escalas específicas donde, a través de experimentos, pueden hacerse y confrontarse hipótesis, obteniendo conclusiones sobre el funcionamiento de los mecanismos estudiados. Esta tesis doctoral se ha enfocado sobre esta problemática intentando mejorar las metodologías del estado del arte y con un objetivo específico: estudiar patrones de deformación que emergen del movimiento organizado de las células durante diferentes estados del desarrollo del embrión, de manera global o en tejidos concretos. Estudios se han centrado en la mecánica en relación con procesos de señalización o interacciones a nivel celular o de tejido. En este trabajo, se propone un esquema para generalizar el estudio del movimiento y las interacciones mecánicas que se desprenden del mismo a diferentes escalas espaciales y temporales. Esto permitiría no sólo estudios locales, si no estudios sistemáticos de las escalas de interacción mecánica dentro de un embrión. Por tanto, el esquema propuesto obvia las causas de generación de movimiento (fuerzas) y se centra en la cuantificación de la cinemática (deformación y esfuerzos) a partir de imágenes de forma no invasiva. Hoy en día las dificultades experimentales y metodológicas y la complejidad de los sistemas biológicos impiden una descripción mecánica completa de manera sistemática. Sin embargo, patrones de deformación muestran el resultado de diferentes factores mecánicos en interacción con otros elementos dando lugar a una organización mecánica, necesaria para el desarrollo, que puede ser cuantificado a partir de la metodología propuesta en esta tesis. La metodología asume un medio continuo descrito de forma Lagrangiana (en función de las trayectorias de puntos materiales que se mueven en el sistema en lugar de puntos espaciales) de la dinámica del movimiento, estimado a partir de las imágenes mediante métodos de seguimiento de células o de técnicas de registro de imagen. Gracias a este esquema es posible describir la deformación instantánea y acumulada respecto a un estado inicial para cualquier dominio del embrión. La aplicación de esta metodología a imágenes 3D + t del pez zebra sirvió para desvelar estructuras mecánicas que tienden a estabilizarse a lo largo del tiempo en dicho embrión, y que se organizan a una escala semejante al del mapa de diferenciación celular y con indicios de correlación con patrones de expresión genética. También se aplicó la metodología al estudio del tejido amnioserosa de la Drosophila (mosca de la fruta) durante el cierre dorsal, obteniendo indicios de un acoplamiento entre escalas subcelulares, celulares y supracelulares, que genera patrones complejos en respuesta a la fuerza generada por los esqueletos de acto-myosina. En definitiva, esta tesis doctoral propone una estrategia novedosa de análisis de la dinámica celular multi-escala que permite cuantificar patrones de manera inmediata y que además ofrece una representación que reconstruye la evolución de los procesos como los ven las células, en lugar de como son observados desde el microscopio. Esta metodología por tanto permite nuevas formas de análisis y comparación de embriones y tejidos durante la embriogénesis a partir de imágenes in-vivo. ABSTRACT The embryogenesis is the process from which a single cell turns into a living organism. Through several stages of development, the cell population proliferates at the same time the embryo shapes and the organs develop gaining their functionality. This is possible through genetic, biochemical and mechanical factors that are involved in a complex interaction of processes organized in different levels and in different spatio-temporal scales. The embryogenesis, through this complexity, develops in a robust and reproducible way, but allowing variability that makes possible the diversity of living specimens. The advances in physics of microscopes and the appearance of fluorescent proteins that can be attached to expression chains, reporting about structural and functional elements of the cell, have enabled for the in-vivo observation of embryogenesis. The imaging process results in sequences of high spatio-temporal resolution 3D+time data of the embryogenesis as a digital representation of the embryos that can be further analyzed, provided new image processing and data analysis techniques are developed. One of the most relevant and challenging lines of research in the field is the quantification of the mechanical factors and processes involved in the shaping process of the embryo and their interactions with other embryogenesis factors such as genetics. Due to the complexity of the processes, studies have focused on specific problems and scales controlled in the experiments, posing and testing hypothesis to gain new biological insight. However, methodologies are often difficult to be exported to study other biological phenomena or specimens. This PhD Thesis is framed within this paradigm of research and tries to propose a systematic methodology to quantify the emergent deformation patterns from the motion estimated in in-vivo images of embryogenesis. Thanks to this strategy it would be possible to quantify not only local mechanisms, but to discover and characterize the scales of mechanical organization within the embryo. The framework focuses on the quantification of the motion kinematics (deformation and strains), neglecting the causes of the motion (forces), from images in a non-invasive way. Experimental and methodological challenges hamper the quantification of exerted forces and the mechanical properties of tissues. However, a descriptive framework of deformation patterns provides valuable insight about the organization and scales of the mechanical interactions, along the embryo development. Such a characterization would help to improve mechanical models and progressively understand the complexity of embryogenesis. This framework relies on a Lagrangian representation of the cell dynamics system based on the trajectories of points moving along the deformation. This approach of analysis enables the reconstruction of the mechanical patterning as experienced by the cells and tissues. Thus, we can build temporal profiles of deformation along stages of development, comprising both the instantaneous events and the cumulative deformation history. The application of this framework to 3D + time data of zebrafish embryogenesis allowed us to discover mechanical profiles that stabilized through time forming structures that organize in a scale comparable to the map of cell differentiation (fate map), and also suggesting correlation with genetic patterns. The framework was also applied to the analysis of the amnioserosa tissue in the drosophila’s dorsal closure, revealing that the oscillatory contraction triggered by the acto-myosin network organized complexly coupling different scales: local force generation foci, cellular morphology control mechanisms and tissue geometrical constraints. In summary, this PhD Thesis proposes a theoretical framework for the analysis of multi-scale cell dynamics that enables to quantify automatically mechanical patterns and also offers a new representation of the embryo dynamics as experienced by cells instead of how the microscope captures instantaneously the processes. Therefore, this framework enables for new strategies of quantitative analysis and comparison between embryos and tissues during embryogenesis from in-vivo images.

Deformable templates for tracking and analysis of intravascular ultrasound sequences

Deformable Template models are first applied to track the inner wall of coronary arteries in intravascular ultrasound sequences, mainly in the assistance to angioplasty surgery. A circular template is used for initializing an elliptical deformable model to track wall deformation when inflating a balloon placed at the tip of the catheter. We define a new energy function for driving the behavior of the template and we test its robustness both in real and synthetic images. Finally we introduce a framework for learning and recognizing spatio-temporal geometric constraints based on Principal Component Analysis (eigenconstraints).

Regional subsidence modelling in Murcia city (SE Spain) using 1-D vertical finite element analysis and 2-D interpolation of ground surface displacements

Subsidence is a hazard that may have natural or anthropogenic origin causing important economic losses. The area of Murcia city (SE Spain) has been affected by subsidence due to groundwater overexploitation since the year 1992. The main observed historical piezometric level declines occurred in the periods 1982–1984, 1992–1995 and 2004–2008 and showed a close correlation with the temporal evolution of ground displacements. Since 2008, the pressure recovery in the aquifer has led to an uplift of the ground surface that has been detected by the extensometers. In the present work an elastic hydro-mechanical finite element code has been used to compute the subsidence time series for 24 geotechnical boreholes, prescribing the measured groundwater table evolution. The achieved results have been compared with the displacements estimated through an advanced DInSAR technique and measured by the extensometers. These spatio-temporal comparisons have showed that, in spite of the limited geomechanical data available, the model has turned out to satisfactorily reproduce the subsidence phenomenon affecting Murcia City. The model will allow the prediction of future induced deformations and the consequences of any piezometric level variation in the study area.

Spatio-temporal scanning and statistical test of the accelerating moment release (AMR) model using Australian earthquake data

The Accelerating Moment Release (AMR) preceding earthquakes with magnitude above 5 in Australia that occurred during the last 20 years was analyzed to test the Critical Point Hypothesis. Twelve earthquakes in the catalog were chosen based on a criterion for the number of nearby events. Results show that seven sequences with numerous events recorded leading up to the main earthquake exhibited accelerating moment release. Two occurred near in time and space to other earthquakes preceded by AM R. The remaining three sequences had very few events in the catalog so the lack of AMR detected in the analysis may be related to catalog incompleteness. Spatio-temporal scanning of AMR parameters shows that 80% of the areas in which AMR occurred experienced large events. In areas of similar background seismicity with no large events, 10 out of 12 cases exhibit no AMR, and two others are false alarms where AMR was observed but no large event followed. The relationship between AMR and Load-Unload Response Ratio (LURR) was studied. Both methods predict similar critical region sizes, however, the critical point time using AMR is slightly earlier than the time of the critical point LURR anomaly.

Dissociating the spatio-temporal characteristics of cortical neuronal activity associated with human volitional swallowing in the healthy adult brain

Human swallowing represents a complex highly coordinated sensorimotor function whose functional neuroanatomy remains incompletely understood. Specifically, previous studies have failed to delineate the temporo-spatial sequence of those cerebral loci active during the differing phases of swallowing. We therefore sought to define the temporal characteristics of cortical activity associated with human swallowing behaviour using a novel application of magnetoencephalography (MEG). In healthy volunteers (n = 8, aged 28-45), 151-channel whole cortex MEG was recorded during the conditions of oral water infusion, volitional wet swallowing (5 ml bolus), tongue thrust or rest. Each condition lasted for 5 s and was repeated 20 times. Synthetic aperture magnetometry (SAM) analysis was performed on each active epoch and compared to rest. Temporal sequencing of brain activations utilised time-frequency wavelet plots of regions selected using virtual electrodes. Following SAM analysis, water infusion preferentially activated the caudolateral sensorimotor cortex, whereas during volitional swallowing and tongue movement, the superior sensorimotor cortex was more strongly active. Time-frequency wavelet analysis indicated that sensory input from the tongue simultaneously activated caudolateral sensorimotor and primary gustatory cortex, which appeared to prime the superior sensory and motor cortical areas, involved in the volitional phase of swallowing. Our data support the existence of a temporal synchrony across the whole cortical swallowing network, with sensory input from the tongue being critical. Thus, the ability to non-invasively image this network, with intra-individual and high temporal resolution, provides new insights into the brain processing of human swallowing. © 2004 Elsevier Inc. All rights reserved.

Spatio-temporal techniques for user identification by means of GPS mobility data

One of the greatest concerns related to the popularity of GPS-enabled devices and applications is the increasing availability of the personal location information generated by them and shared with application and service providers. Moreover, people tend to have regular routines and be characterized by a set of “significant places”, thus making it possible to identify a user from his/her mobility data. In this paper we present a series of techniques for identifying individuals from their GPS movements. More specifically, we study the uniqueness of GPS information for three popular datasets, and we provide a detailed analysis of the discriminatory power of speed, direction and distance of travel. Most importantly, we present a simple yet effective technique for the identification of users from location information that are not included in the original dataset used for training, thus raising important privacy concerns for the management of location datasets.

Temporal and spatio-temporal regimes of generation of Raman fibre lasers

Temporal dynamics of Raman fibre lasers tend to have very complex nature, owing to great cavity lengths and high nonlinearity, being stochastic on short time scales and quasi-continuous on longer time scales. Generally fibre laser intensity dynamics is represented by one-dimensional time-series, which in case of quasi-continuous wave generation in Raman fibre lasers gives little insight into the processes underlying the operation of a laser. New methods of analysis and data representation could help to uncover the underlying physical processes, understand the dynamics or improve the performance of the system. Using intrinsic periodicity of laser radiation, one dimensional intensity time series of a Raman fibre laser was analysed over fast and slow variation time. This allowed to experimentally observe various spatio-temporal regimes of generation, such as laminar, turbulent, partial mode-lock, as well as transitions between them and identify the mechanisms responsible for the transitions. Great cavity length and high nonlinearity also make it difficult to achieve stable high repetition rate mode-locking in Raman fibre lasers. Using Faraday parametric instability in extremely simple linear cavity experimental configuration, a very high order harmonic mode-locking was achieved in ò.ò kmlong Raman fibre laser. The maximum achieved pulse repetition rate was 12 GHz, with 7.3 ps long Gaussian shaped pulses. There is a new type of random lasers – random distributed feedback Raman fibre laser, which temporal properties cannot be controlled by conventionalmode-locking or Q-switch techniques and mechanisms. By adjusting the pump configuration, a very stable pulsed operation of random distributed feedback Raman fibre laser was achieved. Pulse duration varied in the range from 50 to 200 μs depending on the pump power and the cavity length. Pulse repetition rate scaling on the parameters of the system was experimentally identified.