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.

Echo-delay resolution in sonar images of the big brown bat, Eptesicus fuscus

Echolocating big brown bats (Eptesicus fuscus) broadcast ultrasonic frequency-modulated (FM) biosonar sounds (20–100 kHz frequencies; 10–50 μs periods) and perceive target range from echo delay. Knowing the acuity for delay resolution is essential to understand how bats process echoes because they perceive target shape and texture from the delay separation of multiple reflections. Bats can separately perceive the delays of two concurrent electronically generated echoes arriving as little as 2 μs apart, thus resolving reflecting points as close together as 0.3 mm in range (two-point threshold). This two-point resolution is roughly five times smaller than the shortest periods in the bat’s sounds. Because the bat’s broadcasts are 2,000–4,500 μs long, the echoes themselves overlap and interfere with each other, to merge together into a single sound whose spectrum is shaped by their mutual interference depending on the size of the time separation. To separately perceive the delays of overlapping echoes, the bat has to recover information about their very small delay separation that was transferred into the spectrum when the two echoes interfered with each other, thus explicitly reconstructing the range profile of targets from the echo spectrum. However, the bat’s 2-μs resolution limit is so short that the available spectral cues are extremely limited. Resolution of delay seems overly sharp just for interception of flying insects, which suggests that the bat’s biosonar images are of higher quality to suit a wider variety of orientation tasks, and that biosonar echo processing is correspondingly more sophisticated than has been suspected.

Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem–loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I.

We have devised a microspectroscopic strategy for assessing the intracellular (re)distribution and the integrity of the primary structure of proteins involved in signal transduction. The purified proteins are fluorescent-labeled in vitro and reintroduced into the living cell. The localization and molecular state of fluorescent-labeled protein kinase C beta I isozyme were assessed by a combination of quantitative confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and novel determinations of fluorescence resonance energy transfer based on photobleaching digital imaging microscopy. The intensity and fluorescence resonance energy transfer efficiency images demonstrate the rapid nuclear translocation and ensuing fragmentation of protein kinase C beta I in BALB/c3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments.

The extraordinarily rapid disappearance of entoptic images.

It has been known for more than 40 years that images fade from perception when they are kept at the same position on the retina by abrogating eye movements. Although aspects of this phenomenon were described earlier, the use of close-fitting contact lenses in the 1950s made possible a series of detailed observations on eye movements and visual continuity. In the intervening decades, many investigators have studied the role of image motion on visual perception. Although several controversies remain, it is clear that images deteriorate and in some cases disappear following stabilization; eye movements are, therefore, essential to sustained exoptic vision. The time course of image degradation has generally been reported to be a few seconds to a minute or more, depending upon the conditions. Here we show that images of entoptic vascular shadows can disappear in less than 80 msec. The rapid vanishing of these images implies an active mechanism of image erasure and creation as the basis of normal visual processing.

Topological SLAM using a graph-matching based method on omnidirectional images

Comunicación presentada en el XI Workshop of Physical Agents, Valencia, 9-10 septiembre 2010.

An algorithm to hide information in binary images

The objective of this paper is to develop a method to hide information inside a binary image. An algorithm to embed data in scanned text or figures is proposed, based on the detection of suitable pixels, which verify some conditions in order to be not detected. In broad terms, the algorithm locates those pixels placed at the contours of the figures or in those areas where some scattering of the two colors can be found. The hidden information is independent from the values of the pixels where this information is embedded. Notice that, depending on the sequence of bits to be hidden, around half of the used pixels to keep bits of data will not be modified. The other basic characteristic of the proposed scheme is that it is necessary to take into consideration the bits that are modified, in order to perform the recovering process of the information, which consists on recovering the sequence of bits placed in the proper positions. An application to banking sector is proposed for hidding some information in signatures.

A system to communicate hidden information using color images

The objective of this paper is to present a system to communicate hidden information among different users by means of images. The tasks that the system is able to carry on can be divided in two different groups of utilities, implemented in java. The first group of utilities are related with the possibility to hide information in color images, using a steganographic function based on the least significant bit (LSB) methods. The second group of utilities allows us to communicate with other users with the aim to send or receive images, where some information have been previously embedded. Thus, this is the most significant characteristic of the implementation, we have built an environment where we join the email capabilities to send and receive text and images as attached files, with the main objective of hiding information.

Trajectory-Based Morphological Operators: A Model for Efficient Image Processing

Mathematical morphology has been an area of intensive research over the last few years. Although many remarkable advances have been achieved throughout these years, there is still a great interest in accelerating morphological operations in order for them to be implemented in real-time systems. In this work, we present a new model for computing mathematical morphology operations, the so-called morphological trajectory model (MTM), in which a morphological filter will be divided into a sequence of basic operations. Then, a trajectory-based morphological operation (such as dilation, and erosion) is defined as the set of points resulting from the ordered application of the instant basic operations. The MTM approach allows working with different structuring elements, such as disks, and from the experiments, it can be extracted that our method is independent of the structuring element size and can be easily applied to industrial systems and high-resolution images.

3D reconstruction of medical images from slices automatically landmarked with growing neural models

In this study, we utilise a novel approach to segment out the ventricular system in a series of high resolution T1-weighted MR images. We present a brain ventricles fast reconstruction method. The method is based on the processing of brain sections and establishing a fixed number of landmarks onto those sections to reconstruct the ventricles 3D surface. Automated landmark extraction is accomplished through the use of the self-organising network, the growing neural gas (GNG), which is able to topographically map the low dimensionality of the network to the high dimensionality of the contour manifold without requiring a priori knowledge of the input space structure. Moreover, our GNG landmark method is tolerant to noise and eliminates outliers. Our method accelerates the classical surface reconstruction and filtering processes. The proposed method offers higher accuracy compared to methods with similar efficiency as Voxel Grid.

Adjustable compression method for still JPEG images

There are a large number of image processing applications that work with different performance requirements and available resources. Recent advances in image compression focus on reducing image size and processing time, but offer no real-time solutions for providing time/quality flexibility of the resulting image, such as using them to transmit the image contents of web pages. In this paper we propose a method for encoding still images based on the JPEG standard that allows the compression/decompression time cost and image quality to be adjusted to the needs of each application and to the bandwidth conditions of the network. The real-time control is based on a collection of adjustable parameters relating both to aspects of implementation and to the hardware with which the algorithm is processed. The proposed encoding system is evaluated in terms of compression ratio, processing delay and quality of the compressed image when compared with the standard method.

Thermal infrared image processing to assess heat generated by magnetic nanoparticles for hyperthermia applications

Magnetic fluid hyperthermia (MFH) is considered a promising therapeutic technique for the treatment of cancer cells, in which magnetic nanoparticles (MNPs) with superparamagnetic behavior generate mild-temperatures under an AC magnetic field to selectively destroy the abnormal cancer cells, in detriment of the healthy ones. However, the poor heating efficiency of most NMPs and the imprecise experimental determination of the temperature field during the treatment, are two of the majors drawbacks for its clinical advance. Thus, in this work, different MNPs were developed and tested under an AC magnetic field (~1.10 kA/m and 200 kHz), and the heat generated by them was assessed by an infrared camera. The resulting thermal images were processed in MATLAB after the thermographic calibration of the infrared camera. The results show the potential to use this thermal technique for the improvement and advance of MFH as a clinical therapy.

Recursive filtering of images with symmetric extension

Recursive filters are widely used in image analysis due to their efficiency and simple implementation. However these filters have an initialisation problem which either produces unusable results near the image boundaries or requires costly approximate solutions such as extending the boundary manually. In this paper, we describe a method for the recursive filtering of symmetrically extended images for filters with symmetric denominator. We begin with an analysis of symmetric extensions and their effect on non-recursive filtering operators. Based on the non-recursive case, we derive a formulation of recursive filtering on symmetric domains as a linear but spatially varying implicit operator. We then give an efficient method for decomposing and solving the linear implicit system, along with a proof that this decomposition always exists. This decomposition needs to be performed only once for each dimension of the image. This yields a filtering which is both stable and consistent with the ideal infinite extension. The filter is efficient, requiring less computation than the standard recursive filtering. We give experimental evidence to verify these claims. (c) 2005 Elsevier B.V. All rights reserved.

ICICLE: A semantic-based retrieval system for WWW images

In this paper, we present ICICLE (Image ChainNet and Incremental Clustering Engine), a prototype system that we have developed to efficiently and effectively retrieve WWW images based on image semantics. ICICLE has two distinguishing features. First, it employs a novel image representation model called Weight ChainNet to capture the semantics of the image content. A new formula, called list space model, for computing semantic similarities is also introduced. Second, to speed up retrieval, ICICLE employs an incremental clustering mechanism, ICC (Incremental Clustering on ChainNet), to cluster images with similar semantics into the same partition. Each cluster has a summary representative and all clusters' representatives are further summarized into a balanced and full binary tree structure. We conducted an extensive performance study to evaluate ICICLE. Compared with some recently proposed methods, our results show that ICICLE provides better recall and precision. Our clustering technique ICC facilitates speedy retrieval of images without sacrificing recall and precision significantly.