Towards increased efficiency and automation in fluorescence micrograph analysis based on hand-labeled data

Held CH. Towards increased efficiency and automation in fluorescence micrograph analysis based on hand-labeled data. Bielefeld: Universität Bielefeld; 2013.In the past decade, automation in fluorescence microscopy has strongly increased, particularly in regards to image acquisition and sample preparation, which results in a huge volume of data. The amount of time required for manual assessment of an experiment is hence mainly determined by the amount of time required for data analysis. In addition, manual data analysis is often a task with poor reproducibility and lack of objectivity. Using automated image analysis software, the time required for data analysis can be reduced while quality and reproducibility of the evaluation are improved. Most image analysis approaches are based on a segmentation of the image. By arranging several image processing methods in a so-called segmentation pipeline, and by adjusting all parameters, a broad range of fluorescence image data can be segmented. The drawback of available software tools is the long time required to calibrate the segmentation pipeline for an experiment, particularly for researchers with little knowledge of image processing. As a result, many experiments that could benefit from automated image analysis are still evaluated manually. In order to reduce the amount of time users have to spend in adapting automated image analysis software to their data, research was carried out on a novel image analysis concept based on hand-labeled data. Using this concept, the user is required to provide hand-labeled cells, based on which an efficient combination of image processing methods and their parameterization is automatically calibrated, without further user input. The development of a segmentation pipeline that allows high-quality segmentation of a broad range of fluorescence micrographs in short time poses a challenge. In this work, a three-stage segmentation pipeline consisting of exchangeable preprocessing, figure-ground separation and cell-splitting methods was developed. These methods are mainly based on the state of the art, whereas some of them represent contributions to this status. Discretization of parameters must be performed carefully, as a broad range of fluorescence image data shall be supported. In order to allow calibration of the segmentation pipeline in a short time, discretization with equidistant as well as nonlinear step sizes was implemented. Apart from parameter discretization, quality of the calibration strongly depends on choice of the parameter optimization technique. In order to reduce calibration runtime, exploratory parameter space analysis was performed for different segmentation methods. This experiment showed that parameter spaces are mostly monotonous, but also show several local performance maxima. The comparison of different parameter optimization techniques indicated that the coordinate descent method results in a good parameterization of the segmentation pipeline in a small amount of time. In order to minimize the amount of time spent by the user in calibration of the system, correlation between the number of hand-labeled reference samples and the resulting segmentation performance was investigated. This experiment demonstrates that as few as ten reference samples often result in a good parameterization of the segmentation pipeline. Due to the low number of cells required for automatic calibration of the segmentation pipeline, as well as its short runtime, it can be concluded that the investigated method improves automation and efficiency in fluorescence micrograph analysis

Predicting gene function from images of cells

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2007.Includes bibliographical references (p. 107-118).This dissertation shows that biologically meaningful predictions can be made by analyzing images of cells. In particular, groups of related genes and their biological functions can be predicted using images from large gene-knockdown experiments. Our analysis methods focus on measuring individual cells in images from large gene-knockdown screens, using these measurements to classify cells according to phenotype, and scoring each gene according to how reduction in its expression affects phenotypes. To enable this approach, we introduce methods for correcting biases in cell images, segmenting individual cells in images, modeling the distribution of cells showing a phenotype of interest within a screen, scoring gene knockdowns according to their effect on a phenotype, and using existing biological knowledge to predict the underlying biological meaning of a phenotype and, by extension, the function of the genes that most strongly affect that phenotype. We repeat this analysis for multiple phenotypes, extracting for each a set of genes related through that phenotype, along with predictions for the biology of each phenotype. We apply our methods to a large gene-knockdown screen in human cells, validating it on known phenotypes as well as identifying and characterizing several new cellular phenotypes that have not been previously studied.by Thouis Raymond Jones.Sc.D

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

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

Influenza virus assembly

Influenza A Viren besitzen ein segmentiertes, einzelsträngiges RNA-Genom, welches in Form viraler Ribonukleoprotein (vRNP)-Komplexe verpackt ist. Während das virale Genom im Zellkern repliziert wird, finden Assemblierung und Knospung reifer Viruspartikel an der apikalen Plasmamembran statt. Für die Virusbildung müssen die einzelnen viralen Komponenten hierher gebracht werden. Während intrinsische apikale Signale der viralen Transmembranproteine bekannt sind, sind der zielgerichtete Transport und der Einbau des viralen Genoms in neuentstehende Virionen noch wenig verstanden. In dieser Arbeit wurden potentielle Mechanismen des vRNP-Transportes untersucht, wie die Fähigkeit der vRNPs mit Lipidmembranen zu assoziieren und die intrinsische subzellulären Lokalisation des viralen Nukleoproteins (NP), eines Hauptbestandteils der vRNPs. Es konnte gezeigt werden, dass vRNPs nicht mit Lipidmembranen assoziieren, was mittels Flotation aufgereinigter vRNPs mit Liposomen unterschiedlicher Zusammensetzung untersucht wurde. Die Ergebnisse deuten jedoch darauf hin, dass das virale M1 in der Lage ist, Bindung von vRNPs an negativ-geladene Lipidmembranen zu vermitteln. Subzelluläre Lokalisation von NP wurde des Weiteren durch Expression fluoreszierender NP-Fusionsproteine und Fluoreszenzphotoaktivierung untersucht. Es konnte gezeigt werden, dass NP allein nicht mit zytoplasmatischen Strukturen assoziiert, stattdessen aber umfangreiche Interaktionen im Zellkern eingeht und mit hoher Affinität mit bestimmten Kerndomänen assoziiert, und zwar den Nukleoli sowie kleinen Kerndomänen, welche häufig in der Nähe von Cajal-Körperchen und PML-Körperchen zu finden waren. Schließlich wurde ein experimenteller Ansatz etabliert, welcher erlaubt, den Transport vRNP-ähnlicher Komplexe mittels Fluoreszenzdetektion aufzuzeichnen und Einzelpartikelverfolgungsanalysen durchzuführen. Unterschiedliche Phasen des vRNP-Transportes konnten beobachtet werden und ein 3-Phasen-Transportmodell wird skizziert.Influenza A viruses have a segmented single-stranded RNA genome, which is packed in form of viral ribonucleoprotein (vRNP) complexes. While the viral genome is replicated and transcribed in the host cell nucleus, assembly and budding of mature virus particles take place at the apical plasma membrane. Efficient virus formation requires delivery of all viral components to this site. While intrinsic apical targeting signals of the viral transmembrane proteins have been identified, it still remains poorly understood how the viral genome is transported and targeted into progeny virus particles. In this study, potential targeting mechanisms were investigated like the ability of vRNPs to associate with lipid membranes and the intrinsic ability of the viral nucleoprotein (NP) – which is the major protein component of vRNPs – for subcellular targeting. It could be shown that vRNPs are not able to associate with model membranes in vitro, which was demonstrated by flotation of purified vRNPs with liposomes of different lipid compositions. Results indicated, however, that the matrix protein M1 can mediate binding of vRNPs to negatively charged lipid bilayers. Intrinsic subcellular targeting of NP was further investigated by expression of fluorescent NP fusion protein and fluorescence photoactivation, revealing that NP by itself does not target cytoplasmic structures. It was found to interact extensively with the nuclear compartment instead and to target specific nuclear domains with high affinity, in particular nucleoli and small interchromatin domains that frequently localized in close proximity to Cajal bodies and PML bodies. An experimental approach was finally established that allowed monitoring the transport of vRNP-like complexes in living infected cells by fluorescence detection. It was possible to perform single particle tracking and to describe different stages of vRNP transport between the nucleus and the plasma membrane. A model of three-stage transport is suggested

The Effects of Oncogenic Ras on Epithelial Cell Division

The devastating diagnosis of cancer arises in part from the deregulation of cell division. However, it is not known how these cells divide across the range of environments encountered during cancer progression. In this thesis, I investigate the effects of oncogenic Ras on the biology of epithelial cell division. Through this work I find that Ras-ERK signalling not only increases mitotic rounding but also alters the dynamics and division axis of cells as they divide and respread. This reduces the ability of nascent daughters to assume the mother cell footprint and appears to result from effects on cell adhesion that normally provide a memory of cell shape. Significantly, these effects occur within five hours of oncogene expression and are rapidly inhibited by Ras-ERK inhibitors. Together, these findings show how activation of a single oncogene can directly change the fundamental act of cell division as a relatively early event in oncogenesis

Dynamics and interactions of nuclear proteins revealed by quantitative photobleaching microscopy

Tese de doutoramento em Biofísica (Biofísica), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007The nucleus is a complex cellular organelle, exhibiting a high degree of organization and also a highly dynamic nature. Live cell imaging using fluorescent proteins (FPs) as molecular tags and photobleaching techniques have been essential in revealing the dynamic nature of the cell nucleus. In this thesis, these tools were used to study molecular dynamics and interactions inside this cellular compartment. Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) were used to analyze the kinetic behavior of spliceosome components SmE, U2AF65, U2AF35, SF1 and SC35 in the nucleus of living cells. The recruitment mechanism of splicing factors (SFs) to the sites of transcription is still poorly understood. Our results rule out the hypothesis that a transcription specific signal recruits SFs from the speckles. They also suggest the formation of multi-protein complexes distinct from the spliceosome. The existence of these complexes was confirmed by Fluorescence Resonance Energy Transfer (FRET) techniques, which revealed that SFs could interact with each other even in the absence of active splicing. A novel U2AF65 self-interaction was also detected, suggesting altogether that levels of SFs in speckles are consistent with self-organization mechanisms. The intranuclear mobility of mRNPs was studied using two GFP-tagged mRNA-binding proteins, PABPN1 and TAP, as mRNA markers. A novel FLIP method was devised to quantify the mobility of the RNA-bound and unbound pools of molecules and used to test whether myosin motors were implicated in mRNP movement. We show that this is not the case and that myosin inhibition appears to affect transcription instead. A novel FLIP after Photoactivation method was developed to study the nucleocytoplasmic exchange dynamics of nuclear proteins, yielding the permanence times of molecules inside the nucleus. The method was used to study the role of the structural domains of TAP in its shuttling activity.O núcleo celular é um organito complexo, dotado de um elevado grau de organização mas também uma natureza extremamente dinâmica. A utilização de proteínas fluorescentes como marcadores moleculares para visualização em células vivas, bem como as técnicas de photobleaching, têm sido essenciais na descoberta da natureza dinâmica do núcleo. Neste trabalho, estas ferramentas foram aplicadas no estudo da dinâmica e interacções moleculares dentro deste compartimento celular. As técnicas de Fluorescence Recovery After Photobleaching (FRAP) e Fluorescence Loss In Photobleaching (FLIP) foram utilizadas na análise do comportamento cinético dos componentes do spliceosoma SmE, U2AF65, U2AF35, SF1 e SC35 no interior do núcleo de células vivas. O mecanismo de recrutamento dos factores de splicing (SFs) para os locais de transcrição é ainda pouco conhecido. Os nossos resultados excluem a hipótese de haver um sinal associado à transcrição que seja responsável por este recrutamento. Sugerem ainda a formação de complexos multi-proteicos distintos do spliceosoma. A existência destes complexos foi confirmada por técnicas de Fluorescence Resonance Energy Transfer (FRET), que mostraram que os SFs podiam interagir uns com os outros mesmo na ausência de splicing activo. Foi ainda descoberta uma nova auto-interacção para o factor U2AF65, sugerindo os resultados no seu conjunto que a distribuição de SFs no núcleo é compatível com mecanismos de auto-organização. A mobilidade de mRNPs no núcleo foi estudada utilizando como marcadores moleculares duas proteínas que se ligam ao mRNA marcadas com GFP, PABPN1 e TAP. Foi desenvolvido um método de FLIP para quantificação da mobilidade das fracções ligadas e não ligadas ao mRNA e usado para testar a possibilidade de motores de miosina estarem envolvidos no movimento de mRNPs. Mostramos que tal não acontece e que a inibição de miosina parece antes afectar a transcrição. Um novo método de FLIP após foto-activação foi desenvolvido para estudar a dinâmica de trocas entre o núcleo e o citoplasma de proteínas nucleares, permitindo a estimação do tempo de permanência de moléculas dentro do núcleo. O método foi utilizado para investigar o papel dos diferentes domínios estruturais da proteína TAP na sua actividade de exportação nuclear.Fundação para a Ciência e Tecnologia (BD/21518/99); European Commission (“RNOMICS” QLG2-CT-2001-01554 and “Integrated Technologies for in vivo Molecular Imaging” LSHG-CT-2003-503259

Electron microscopy of macromolecular complexes, and their interactions with membranes

Over the last ~4 years, cryo-electron microscopy (EM) has undergone a ‘revolution’, thanks to advances in microscope hardware, such as direct electron detectors, and image processing algorithms. This has improved the quality of data that can be obtained using cryo-EM, and so a diverse range of biological problems can now be effectively tackled using this technique. Here, two biological systems are examined using different cryo-EM imaging modalities. A large number of human diseases are associated with the formation of amyloid fibrils. Amyloid-membrane interactions may play a key role in amyloid mediated cytotoxicity. In Chapter Three, a combination of liposome dye release assays and cryo-EM is used to investigate the effect of amyloid fibrils on membranes of varying compositions. Solution conditions such as pH, alongside lipid composition, were found to have a profound effect on the propensity of β-2-microglobulin (β2m) amyloid fibrils to perturb membranes. In Chapter Four, subcellular fractionation and cryo-EM and tomography were used to further probe the nature of interactions between β2m amyloid fibrils and cellular membranes. Cryo-electron tomography was used to reveal 3D detail of unique interactions at molecular resolution. In Chapter Five, structural properties of the Leviviridae family of bacteriophages, a model family of spherical viruses, were investigated using cryo-EM. The structure of Qβ bacteriophage at 4.2 Å is presented. Using this electron density map, an existing X-ray crystal structure is refined to yield a better quality model of the Qβ capsid. Asymmetric reconstructions were used to generate insight into genome organisation and capsid assembly. This work demonstrates the utility of cryo-EM as a flexible technique to tackle a broad range of research questions by providing structural information at different resolutions

Combining active contours and active shapes for segmentation of fluorescently stained cells: Application to virology

Fluorescence microscopy is an essential tool to examine host-pathogen interactions such as the influence of Fascin on cell-cell contacts between infected and uninfected cells. Manual analysis of fluorescence microscopy images is prone to errors leading to inter- and intra-observer variability. To increase reproducibility and objectivity, automated and semi-automated image processing methods are required. For a reliable segmentation of touching and overlapping cells, we propose an active contours algorithm extended by an energy term based on an active shape model. The algorithm is evaluated on confocal cell image data labeled by a human expert

MC 2019 Berlin Microscopy Conference - Abstracts

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2019", die vom 01. bis 05.09.2019, in Berlin stattfand

Modeling and quantitative analysis of actin cytoskeleton networks

In eukaryotischen Zellen bildet das Strukturprotein Aktin Polymernetzwerke aus, die sehr dynamisch und für viele zelluläre Prozesse lebenswichtig sind. In dieser Arbeit werden theoretische Konzepte vorgestellt, um die Eigenschaften komplexer Aktin-Netzwerkstrukturen zu verstehen und mit Messungen mittels Fluoreszenz- und Elektronenmikroskopie zu vergleichen. Ein Großteil der Arbeit behandelt dabei flache vernetzte Aktinstrukturen, die durch gerichtete Polymerisation gegen eine äußere Kraft anwachsen. Dieser Netzwerktyp ist ein wichtiger Bestandteil von sich bewegenden Zellen, wird aber auch von intrazellulären Pathogenen zur Fortbewegung missbraucht. Eine zentrale, experimentell messbare Eigenschaft solcher Netzwerke ist ihre Kraft-Geschwindigkeits-Relation. Verschiedene aktuelle Messungen ergaben hierfür widersprüchlich erscheinende Ergebnisse. In einem relativ einfachen physikalischen Modell wird gezeigt, dass in wachsenden Aktin-Netzwerken zwei stationäre Filament-Orientierungsverteilungen miteinander konkurrieren. Strukturelle Übergänge zwischen den beiden Architekturen werden durch Änderung der Wachstumsgeschwindigkeit des Netzwerks initiiert. Mit zusätzlichen Annahmen zur mechanischen Stabilität einzelner Filamente werden die experimentell gefundenen Eigenarten der Kraft-Geschwindigkeits-Relation (eine Abfolge von konvexen und konkaven Verläufen sowie Hysterese) theoretisch begründet. Das Modell wird zusätzlich auf Aktinwachstum gegen gekrümmte Hindernisse wie intrazelluläre Pathogene erweitert. Um in der Zukunft spezifische Vorhersagen des Modells experimentell zu überprüfen, wurde eine Methode zur automatischen Analyse von Elektronenmikroskopiebildern von Aktin-Netzwerken entwickelt. Erste Ergebnisse lassen eine gute Übereinstimmung erwarten. Des Weiteren wurde eine Methode entwickelt, um Änderungen in der Aktin-Struktur von adhärenten Zellen in einem Hochdurchsatzverfahren mit Fluoreszenzmikroskopie zu bewerten