87 research outputs found

    TOMOBFLOW: feature-preserving noise filtering for electron tomography

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    <p>Abstract</p> <p>Background</p> <p>Noise filtering techniques are needed in electron tomography to allow proper interpretation of datasets. The standard linear filtering techniques are characterized by a tradeoff between the amount of reduced noise and the blurring of the features of interest. On the other hand, sophisticated anisotropic nonlinear filtering techniques allow noise reduction with good preservation of structures. However, these techniques are computationally intensive and are difficult to be tuned to the problem at hand.</p> <p>Results</p> <p>TOMOBFLOW is a program for noise filtering with capabilities of preservation of biologically relevant information. It is an efficient implementation of the Beltrami flow, a nonlinear filtering method that locally tunes the strength of the smoothing according to an edge indicator based on geometry properties. The fact that this method does not have free parameters hard to be tuned makes TOMOBFLOW a user-friendly filtering program equipped with the power of diffusion-based filtering methods. Furthermore, TOMOBFLOW is provided with abilities to deal with different types and formats of images in order to make it useful for electron tomography in particular and bioimaging in general.</p> <p>Conclusion</p> <p>TOMOBFLOW allows efficient noise filtering of bioimaging datasets with preservation of the features of interest, thereby yielding data better suited for post-processing, visualization and interpretation. It is available at the web site <url>http://www.ual.es/%7ejjfdez/SW/tomobflow.html</url>.</p

    Mathematical Methods for the Quantification of Actin-Filaments in Microscopic Images

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    In cell biology confocal laser scanning microscopic images of the actin filament of human osteoblasts are produced to assess the cell development. This thesis aims at an advanced approach for accurate quantitative measurements about the morphology of the bright-ridge set of these microscopic images and thus about the actin filament. Therefore automatic preprocessing, tagging and quantification interplay to approximate the capabilities of the human observer to intuitively recognize the filaments correctly. Numerical experiments with random models confirm the accuracy of this approach

    Mathematical Morphology for Quantification in Biological & Medical Image Analysis

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    Mathematical morphology is an established field of image processing first introduced as an application of set and lattice theories. Originally used to characterise particle distributions, mathematical morphology has gone on to be a core tool required for such important analysis methods as skeletonisation and the watershed transform. In this thesis, I introduce a selection of new image analysis techniques based on mathematical morphology. Utilising assumptions of shape, I propose a new approach for the enhancement of vessel-like objects in images: the bowler-hat transform. Built upon morphological operations, this approach is successful at challenges such as junctions and robust against noise. The bowler-hat transform is shown to give better results than competitor methods on challenging data such as retinal/fundus imagery. Building further on morphological operations, I introduce two novel methods for particle and blob detection. The first of which is developed in the context of colocalisation, a standard biological assay, and the second, which is based on Hilbert-Edge Detection And Ranging (HEDAR), with regard to nuclei detection and counting in fluorescent microscopy. These methods are shown to produce accurate and informative results for sub-pixel and supra-pixel object counting in complex and noisy biological scenarios. I propose a new approach for the automated extraction and measurement of object thickness for intricate and complicated vessels, such as brain vascular in medical images. This pipeline depends on two key technologies: semi-automated segmentation by advanced level-set methods and automatic thickness calculation based on morphological operations. This approach is validated and results demonstrating the broad range of challenges posed by these images and the possible limitations of this pipeline are shown. This thesis represents a significant contribution to the field of image processing using mathematical morphology and the methods within are transferable to a range of complex challenges present across biomedical image analysis

    Optical microscopy to study the role of cytoskeleton in cell locomotion and virus trafficking

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    3. General conclusions 150 The interest in optical microscopy is constanly growing, mainly because of its unique features in examining biological systems in four dimensions (x-y-z-t)1 . The work presented here was focused on biological applications of optical microscopy by exploring and improving the spatial and temporal resolution performances and by futher developing optical tools for manipulating biological samples. First, I studied the resolution performances of the system in the three dimensional space and I contributed in improving the experimental spatial resolution of microscope by applying deconvolution. In this respect, theoretical modelling can characterize the image formation process of the microscope, but only experimental measurement of the PSF can quantify the limitations of the real system. Indeed, experimental PSF presents shape assymetry due to spherical aberrations introduced by optical elements, while theoretical PSF is symmetric and account only for the resolution limits of an ideal imaging system. The disadvantage of experimental PSF is that could be corrupted by noise, otherwise deconvolution with the theoretical PSF offer only a qualitative improvement of the image, because the introduced artefacts cannot be quantified. Deconvolution of the acquired data with experimental PSF...3. General conclusions 150 The interest in optical microscopy is constanly growing, mainly because of its unique features in examining biological systems in four dimensions (x-y-z-t)1 . The work presented here was focused on biological applications of optical microscopy by exploring and improving the spatial and temporal resolution performances and by futher developing optical tools for manipulating biological samples. First, I studied the resolution performances of the system in the three dimensional space and I contributed in improving the experimental spatial resolution of microscope by applying deconvolution. In this respect, theoretical modelling can characterize the image formation process of the microscope, but only experimental measurement of the PSF can quantify the limitations of the real system. Indeed, experimental PSF presents shape assymetry due to spherical aberrations introduced by optical elements, while theoretical PSF is symmetric and account only for the resolution limits of an ideal imaging system. The disadvantage of experimental PSF is that could be corrupted by noise, otherwise deconvolution with the theoretical PSF offer only a qualitative improvement of the image, because the introduced artefacts cannot be quantified. Deconvolution of the acquired data with experimental PSF...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

    Locality sensitive modelling approach for object detection, tracking and segmentation in biomedical images

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    Biomedical imaging techniques play an important role in visualisation of e.g., biological structures, tissues, diseases and medical conditions in cellular level. The techniques bring us enormous image datasets for studying biological processes, clinical diagnosis and medical analysis. Thanks to recent advances in computer technology and hardware, automatic analysis of biomedical images becomes more feasible and popular. Although computer scientists have made a great effort in developing advanced imaging processing algorithms, many problems regarding object analysis still remain unsolved due to the diversity of biomedical imaging. In this thesis, we focus on developing object analysis solutions for two entirely different biomedical image types: uorescence microscopy sequences and endometrial histology images. In uorescence microscopy, our task is to track massive uorescent spots with similar appearances and complicated motion pattern in noisy environments over hundreds of frames. In endometrial histology, we are challenged by detecting different types of cells with similar appearance and in terms of colour and morphology. The proposed solutions utilise several novel locality sensitive models which can extract spatial or/and temporal relational features of the objects, i.e., local neighbouring objects exhibiting certain structures or patterns, for overcoming the difficulties of object analysis in uorescence microscopy and endometrial histology

    Particle Filtering Methods for Subcellular Motion Analysis

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    Advances in fluorescent probing and microscopic imaging technology have revolutionized biology in the past decade and have opened the door for studying subcellular dynamical processes. However, accurate and reproducible methods for processing and analyzing the images acquired for such studies are still lacking. Since manual image analysis is time consuming, potentially inaccurate, and poorly reproducible, many biologically highly relevant questions are either left unaddressed, or are answered with great uncertainty. The subject of this thesis is particle filtering methods and their application for multiple object tracking in different biological imaging applications. Particle filtering is a technique for implementing recursive Bayesian filtering by Monte Carlo sampling. A fundamental concept behind the Bayesian approach for performing inference is the possibility to encode the information about the imaging system, possible noise sources, and the system dynamics in terms of probability density functions. In this thesis, a set of novel PF based metho

    Development of polarization-resolved optical scanning microscopy imaging techniques to study biomolecular organizations

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    Light, as electromagnetic radiation, conveys energy through space and time via fluctuations in electric and magnetic fields. This thesis explores the interaction of light and biological structures through polarization-resolved imaging techniques. Light microscopy, and polarization analysis enable the examination of biological entities. Biological function often centers on chromatin, the genetic material composed of DNA wrapped around histone proteins within cell nuclei. This structure's chiral nature gives rise to interactions with polarized light. This research encompasses three main aspects. Firstly, an existing multimodal Circular Intensity Differential Scattering (CIDS) and fluorescence microscopy are upgraded into an open configuration to be integrated with other modalities. Secondly, a novel cell classification method employing CIDS and a phasor representation is introduced. Thirdly, polarization analysis of fluorescence emission is employed for pathological investigations. Accordingly, the thesis is organized into three chapters. Chapter 1 lays the theoretical foundation for light propagation and polarization, outlining the Jones and Stokes-Mueller formalisms. The interaction between light and optical elements, transmission, and reflection processes are discussed. Polarized light's ability to reveal image contrast in polarizing microscopes, linear and nonlinear polarization-resolved microscopy, and Mueller matrix microscopy as a comprehensive technique for studying biological structures are detailed. Chapter 2 focuses on CIDS, a label-free light scattering method, including a single point angular spectroscopy mode and scanning microscopy imaging. A significant upgrade of the setup is achieved, incorporating automation, calibration, and statistical analysis routines. An intuitive phasor approach is proposed, enabling image segmentation, cell discrimination, and enhanced interpretation of polarimetric contrast. As a result, image processing programs have been developed to provide automated measurements using polarization-resolved laser scanning microscopy imaging integrated with confocal fluorescence microscopy of cells and chromatin inside cell nuclei, including the use of new types of samples such as progeria cells. Chapter 3 applies a polarization-resolved two-photon excitation fluorescence (2PEF) microscopy to study multicellular cancerous cells. A homemade 2PEF microscope is developed for colon cancer cell analysis. The integration of polarization and fluorescence techniques leads to a comprehensive understanding of the molecular orientation within samples, particularly useful for cancer diagnosis. Overall, this thesis presents an exploration of polarization-resolved imaging techniques for studying biological structures, encompassing theory, experimental enhancements, innovative methodologies, and practical applications

    Active nematic turbulence: An experimental study

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    [eng] One of the most striking phenomena of active fluids, i.e., fluids composed of self-propelled constituents, is the emergence of chaotic spatiotemporal flows. "This regime, reminiscent of inertial turbulence but happening at low Reynolds numbers, has become to be known as active turbulence. It has been observed in a variety of systems, such as bacterial suspensions or epithelial tissues. Despite the visual similarities, active turbulent flows possess fundamental differences from classical turbulent flows. The differences essentially emanate from the fact that active turbulence originates at vanishing Reynolds numbers from the self-organization of the fluid constituents, which move coordinately at distances much larger than their own size. As a result, active chaotic flows are endowed with a characteristic length scale. In this thesis, working with an experimental active system displaying nematic order, i.e., head-to- tail orientational order, and composed of proteins from the cytoskeleton, we address some still- standing open questions regarding active turbulence. More specifically, since our experimental system is two-dimensional and has nematic order, we study 2D active nematic turbulence. We begin this thesis by unveiling the pathway followed by the active fluid with an imposed radial alignment to its final characteristic chaotic state. More in particular, we demonstrate that the AN in the aster configuration is intrinsically unstable to buckling. In turn, a characteristic length scale already emerges at the instability's early stages. Interestingly, the instability of the aligned active nematic can be characterized in terms of a growth rate that exhibits a quadratic or quasi-quadratic dependence on the leading wavenumber. Our experimental results are then compared with predictions obtained from linear stability analysis. This enables us to see that the coupling of the active nematic with adjacent fluid layers precludes long wavelength fluctuations, imposing in this way a genuine wavelength selection mechanism. In the second project, we measure the active liquid crystal's flow field and the associated kinetic energy spectrum. In this way, we verify the existence of scaling regimes, some of which feature exponents previously predicted through theory and simulations , together with new ones. To understand the newly-discovered scaling regimes, we exploit a theory that includes the hydrodynamic coupling of the active nematic with the two contacting passive fluid layers. This theory assesses the range of validity of the identified scaling regimes, and permits to extract information on important rheological parameters of the active fluid In the final project, still in progress, we address the presence of energy cascades in active nematic turbulence. Preliminary experimental results, supported with simulations, suggest that even though the free energy balance does not entirely vanish at all length scales, we cannot indeed conclude that there is energy transfer between scales. A significant limitation we encounter when computing the free energy balance of the active nematic is that most of the material parameters still need to be determined. Therefore, further research research devoted to the evaluation of such parameters may shed light on this respect. On top of the above fundamental studies, we also demonstrate two implementations of polarimetry measurements coupled with fluorescence imaging, with which we can simultaneously measure the director and velocity fields of the active nematic . The first arrangement is based on a liquid crystal slab, whose retardance can be easily commanded with a computer. By measuring the light intensity reaching the detector at different configurations of the liquid crystal retarder, we can unequivocally and continuously know the sample's local orientation. The alternative implementation incorporates a polarization camera, a device composed of subpixels with different polarizations. This arrangement allows us to obtain exceptional birefringence measurements at significantly high frame rates, even with very low-birefringent samples, as the active nematic.[cat] Els fluids actius, com les suspensions de bacteris o els teixits, són fluids compostos per moltes unitats capaces de propulsar-se contínuament. Aquests fluids presenten propietats molt interessants i radicalment oposades a les que s’observen quan una d’aquestes unitats actives es mou individualment. Un exemple és el que es coneix com a turbulència activa, on els fluids actius es mouen caòticament i que emergeix inclús a números de Reynolds baixos, quan la inèrcia es menyspreable. Aquest fenomen es diu així perquè visualment recorda a la turbulència inercial clàssica. Tot i així, hi ha diferències fonamentals entre aquests dos tipus de turbulència, els quals ens interessa discernir. En aquesta tesi es presenten estudis experimentals, duts a terme amb una suspensió de proteïnes del citoesquelet, i amb els quals abordem algunes qüestions encara obertes sobre la turbulència activa i les seves similituds i diferències amb la turbulència inercial. Més concretament, com el nostre sistema experimental és 2D i presenta simetria nemàtica, nosaltres estudiem la turbulència nemàtica activa. En primer lloc, revelem el camí que segueix el nemàtic actiu alineat radialment fins que arriba al seu estat turbulent. Més concretament, demostrem que la geometria d’àster és inestable; en conseqüència, el fluid actiu comença a deformar-se i llavors emergeix una escala de longitud característica. Després duem a terme estudis on l’actiu nemàtic ja es troba plenament en el seu estat turbulent. En aquest sentit, demostrem l’existència de règims d’escala en l’espectre d’energia cinètica del nemàtic actiu i abordem la presència o no presència de cascades d’energia en el context de la turbulència nemàtica activa. Finalment, descrivim dues tècniques de polarimetria acoblades a fluorescència amb les quals podem mesurar simultàniament la orientació i el camp de velocitats del nemàtic actiu i que ens permeten dur a terme les mesures experimentals presentades en aquesta tesi
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