Quantitative Imaging in Electron and Confocal Microscopies for Applications in Biology

Among the large number of topics related to the quantification of images in electron and confocal microscopies for applications in biology, we selected four subjects that we consider to be representative of some recent tendencies. The first is the quantification of three-dimensional data sets recorded routinely in scanning confocal microscopy. The second is the quantification of the textural and fractal appearance of images. The two other topics are related to image series, which are more and more often provided by imaging instruments. The first kind of series concerns electron energy-filtered images. We show that the parametric (modelling) approach can be complemented by non-parametric approaches (e.g., different variants of multivariate statistical techniques). The other kind of series consists of multiple mappings of a specimen. We describe several new tools for the study and quantification of the co-location, with potential application to multiple mappings in microanalysis or in fluorescence microscopy

Quantitative Analysis of Chromosome/Gene Spatial Distribution

Zusammenfassung Die Fortschritte in den Bereichen Zellbildgebung und Mikroskopie haben eine intensive Untersuchung der räumlichen Anordnung von Chromosomen bzw. Genen im Verlauf der letzten Jahre möglich gemacht. Es wurden bereits Algorithmen zur Quantifizierung der räumlichen Anordnung von Chromosomen bzw. Genen entwickelt, wobei die meisten dieser Methoden jedoch auf zweidimensionalen (2D) Bilddaten basieren. Um dreidimensionale (3D) konfokale Bilddaten verarbeiten zu können, ist es notwendig neue Algorithmen zu entwickeln, die auf 3D Datensätzen basieren. In dieser Arbeit werden neue Methoden zur Beschreibung, Analyse und Visualisierung der 3D Verteilung von Chromosomen und Genen in fixierten Zellkernen in 3D Bilddaten präsentiert, die basierend auf Konzepten der objektorientierten Programmierung in der Programmiersprache Java implementiert wurden. Kapitel 2 beschreibt verschiedene Softwarewerkzeuge zur Bestimmung von Ähnlichkeiten der Anordnung von Chromosomen unter Verwendung der Krümmungsenergie von Thin-plate Splines sowie zur Berechnung von geometrischen Mittelpunkten, Distanzen und Winkeln. Zwei anwendungsorientierte Projekte zur räumlichen Verteilung von Chromosomen bzw. Genen werden in Kapitel 3 bzw. Kapitel 4 vorgestellt. Die Vorteile, Grenzen und weitere Verbesserungen von diesen Computermethoden werden ausführlich in Kapitel 5 behandelt. Danach wird ein neues Modell der Vererbung der räumlichen Chromosomenordnung erläutert. Die quantitative Analyse in Kapitel 3 zeigt, dass die Unterschiede in der Anordnung von Chromosomenterritorien kontinuierlich mit der Anzahl der Zellgenerationen (d.h. Zellteilungen) zunehmen. In HeLa Zellklonen sind die Unähnlichkeiten in der Anordnung von Chromosomenterritorien nach fünf oder sechs Zellteilungen bereits so groß wie die zwischen unverwandten, zufällig ausgewählten Zellen. Die quantitative Analyse in Kapitel 4 zeigt, dass während der Interphase die Positionen der untersuchten Gene (MLL und fünf seiner Translokationspartner) sowie von vier chromosomalen Kontroll-Loci ein charakteristisches Verteilungsmuster innerhalb des Zellkerns besitzen. Dies gilt für jede der untersuchten hämatopoietischen Zellen. Die in dieser Arbeit präsentierten Methoden zur Bestimmung der Ähnlichkeit der Anordnung von Chromosomen unter Verwendung von punktbasierter Registrierung liefern zum ersten Mal Beispiele zur Analyse und Bewertung der Vererbung einer räumlichen Verteilung von Chromosomen über mehrere Zellteilungen hinweg. Gleichzeitig wird zum ersten Mal die räumliche Verteilung von Genen, vor allem des Genes MLL und einiger Translokationspartner, im 3D Raum des Interphasezellkerns hämatopoietischer Zellen quantitativ beschrieben

2D mapping of strongly deformable cell nuclei, based on contour matching

The spatiotemporal dynamics of protein complexes and genome loci are functionally linked to cellular health status. To study the inherent motion of subnuclear particles, it is essential to remove any superimposed component stemming from displacement and deformation of the nucleus. In this article, we propose a mapping of the nuclear interior, which is based on the deformation of the nuclear contour and has no shape constraints. This registration procedure enabled an accurate estimation of telomere mobility in living human cells undergoing dramatic nuclear deformations. Given the large variety of pathologies and cellular processes that are associated with strong nuclear shape changes, the contour mapping algorithm has generic value for improving the accuracy of mobility measurements of genome loci and intranuclear macromolecule complexes

Modelling Nuclear Body Dynamics in Living Cells by 4-D Microscopy, Image Analysis and Simulation

The work presented here demonstrates rules of and validates models for nuclear body (NB) dynamics. Simulation tools developed in the course of this work can be used in future work to generate hypotheses about related aspects of nuclear architecture. Initially I examined the mobility of vimentin nuclear bodies bodies (VNB) in interphase by single particle tracking and analysis of fluorescence images from 4-D confocal laser scanning microscopy (CLSM). These synthetic nuclear bodies are observed in cells transfected with labelled nuclear-targeted Xenopus laevis vimentin. Analysis shows that VNBs undergo anomalous diffusion in the nuclei, independent of metabolic energy. Individual bodies display either one of the three modes of diffusion -- directed, restricted or simple. The consistency of modes and magnitudes of diffusion constants between VNBs and bona fide nuclear bodies points to a generic mechanism that mediates and regulates the mobility of nuclear bodies. Since the results of diffusion analysis of VNBs did not agree with a simple diffusion model, I tested the alternative interchromosomal domain (ICD) compartment model. The ICD model predicts that in interphase cell nuclei, individual decompacted chromosomes do not intermingle, but are separated by a significant interchromatin space forming a network of channels. These networks could affect the mobility of nuclear bodies. Monte Carlo simulations that predict the effects of channels and other obstructions on NB diffusion were tested, but they could not explain deviation from ideal behaviour. Fitting an empirical model of `critical diffusion' produced similar results. Therefore the ICD model as a purely obstructing network of channels needs modification, to possibly include binding. To examine the role of chromatin density in intra-nuclear diffusion, I employed multidimensional fluorescence recovery after photobleaching (FRAP) in living cells. The influence of chromatin density on diffusive mobility of the nuclear yellow fluorescent protein (YFP) appears marginal. A 2-D diffusion simulation to better characterize the experiment provides a tool to produce `diffusion maps' of the nucleus. The related aspect of nuclear body integrity and dynamics was examined for the distribution of topoisomerase II beta (TopoIIb), which localizes preferentially in the nucleolus. The experimentally observed diffusion and binding dynamics were formulated as a compartment model and fitted to the experiments. The model topology, flux constants and residence times estimates could be validated, providing a predictive model of TopoIIb dynamics By demonstrating that VNB diffusion is anomalous and consistent with other bona fide NBs, I have revealed a mechanism that regulates NB mobility. The diffusion of these bodies deviates however from ideal diffusion, and can be explained by neither the effect of chromatin density on molecular diffusion, nor the different models of NB diffusion. I have shown that binding rather than diffusion appears to determine nuclear body localization and dynamics, as in the case of TopoIIb. Nuclear bodies and nuclear architecture has been recently hypothesized as emerging from simple local interactions. The predictive model for TopoIIb distribution dynamics provides evidence for this. The models presented here, are in keeping with the increasing trend to abstract nuclear dynamics as mathematical models. It is hoped that the work presented here will contribute to the effort of arriving at an integrated model for nuclear bodies and therefore better understanding nuclear architecture

Rekonstruktion und Quantifizierung von Chromosomendomänen in Interphasezellen

Das Laserscanning-Mikroskop ermöglicht mit seinem verbesserten axialen Auflösungsvermögen die räumliche in-situ Aufnahme subzellulärer Strukturen. Zusammen mit der Fluoreszenz in-situ Hybridisierung sind damit Abbildungen von Chromosomendomänen in Interphasezellen möglich geworden. Die vorliegende Dissertation beschreibt den Ablauf und die Auswertung solcher Messungen. Betrachtet werden zunächst Vorexperimente, die eine Charakterisierung des Mikroskops erlauben. Im wesentlichen wird die Bestimmung des helligkeitsabhängigen Signal/Rausch-Verhältnisses, der Intensitätsübertragung, der chromatischen Abberation und der Punktübertragungsfunktion an digitalisierten Bildern gezeigt. Einen Schwerpunkt der Arbeit bildet die Segmentierung der Domänen, die auf das Vorwissen über den DNA-Gehalt des betrachteten Chromosoms zurückgreift. Gezeigt werden abschließend Methoden der Bildvorverarbeitung, die eine sinnvolle Quantifizierung der Domänen durch ebenfalls demonstrierte Schätzer (Cavalieri, Marching Cubes) erlauben

Role of Plectin in Nuclear Mechano-transduction of Keratinocytes.

PhDCell shape dictates nuclear morphology via cytoskeleton remodelling, potentially regulating therefore nuclear architecture and gene expression. Utilising micro-patterned substrates to control cell shape, the physical cell-nuclear integration was investigated. Cells were seeded in patterns with variable sizes and shapes, and the nuclear morphology assessed. The nucleus in spread cells was larger when compared with confined cells as measured by its volume. Actin and keratins are the two main determinants of keratinocytes mechanics and their role in defining nuclear morphology is still unclear. Results here showed that the inhibition of filamentous actin, therefore the formation of stress fibres, was not sufficient to disrupt the physical link between cell shape and nuclear morphology in keratinocytes. Plectin deficient keratinocytes were further investigated as they possess abnormal keratin architecture: their more open network of keratin 14 resulted in a larger and more deformable nucleus. These deformations depended on acto-myosin contractility and the formation of cell-cell contacts. When seeded as cell clusters with a confined adhesive area, Plec KO were able to compact to a higher extent and when allowed to migrate outside this confined area, the nucleus of Plec KO underwent larger and quicker morphological remodelling in comparison to the WT. Changes in nuclear morphology may influence chromatin remodelling. Confined keratinocytes committed to terminal differentiation, had lower levels of transcriptionally active sites (H3K4me3), and re-distributed heterochromatin markers (H3K27me3). In summary, the present thesis demonstrates that cell shape mediates nuclear morphology and chromatin remodelling, and this cell-nucleus coupling is coordinated by the cross-talk between keratins and actin, in which plectin is a key playe

Spatial organization of transcription as a potential gene expression mechanism in Plasmodium falciparum

Crescentes evidências mostram que a organização espacial da transcrição é um fator epigenético importante na regulação gênica em eucariotos. Em células de mamíferos, os genes são transcritos em estruturas nucleares discretas conhecidas como fábricas de transcrição, e genes com funções relacionadas e co-regulados compartilham as mesmas fábricas de transcrição. Plasmodium falciparum apresenta um padrão de expressão gênica bastante complexo durante o ciclo eritrocítico, contrastando paradoxalmente com o baixo número de putativos fatores de transcrição codificados pelo seu genoma. Por outro lado, mecanismos epigenéticos são importantes na regulação gênica neste parasita. Nesta tese, investigamos a organização da transcrição em P. falciparum visando determinar se a organização nuclear está relacionada com a expressão/regulação gênica ao longo do desenvolvimento do parasita. Com este objetivo, marcamos transcritos nascentes com BrUTP em formas eritrocíticas de P. falciparum. Assim como em mamíferos, a transcrição no parasita também está organizada em focos nucleoplásmicos discretos, chamados fábricas de transcrição. Análises automatizadas de imagens em 3D mostram que o número e a intensidade das fábricas de transcrição variam durante o ciclo eritrocítico, estando correlacionados com o número de genes expressos em cada estágio, mas não com o volume nuclear. O baixo número de fábricas indica que os genes ativos compartilham as fábricas enquanto estão sendo transcritos. Surpreendentemente, as fábricas são espacilamente redistribuídas durante o desenvolvimento de anéis para trofozoítas, com a periferia nuclear sendo a zona de transcrição favorita nos anéis, enquanto nos trofozoítas as fábricas estão igualmente distribuídas por todo o nucleoplasma. Também observamos que a transcrição por RNA polimerase I ocorre principalmente nas áreas centrais dos núcleos em trofozoítas, sugerindo que os componentes nucleolares podem ser dispersos devido à entrada na fase S. Assim como nos eucariotos superiores, as fábricas de transcrição em P. falciparum também se localizam em áreas nucleares com baixa densidade de cromatina. Análises de imunofluorescência combinando incorporação de BrUTP com marcadores nucleares mostram que as fábricas de transcrição formam um compartimento exclusivo, diferente do compartimento de silenciamento definido por PfSir2A ou do compartimento de cromatina ativa definido por H4ac ou H3K79me3. Para estudar a organização espacial da cromatina e entender como os genes co-regulados interagem com as fábricas de transcrição, decidimos realizar ensaios de hibridização fluorescente in situ (fluorescence in situ hybridization, FISH). Durante a realização de ensaios de FISH seguindo protocolos publicados, observamos uma grande variação na morfologia nuclear dos parasitas, o que nos moveu a otimizar esta técnica. Utilizando análises automatizadas de imagens, mostramos que os parasitas desidratados por secagem ao ar e fixados por curtos períodos de tempo à temperatura ambiente apresentam uma variação intra-populacional maior em relação à forma e ao volume nucleares após o ensaio de FISH, assim como valores de volume quase duas vezes maiores, do que núcleos de parasitas fixados em suspensão por longos períodos de tempo e em baixas temperaturas. Também observamos que a fixação em suspensão leva a uma melhor conservação da estrutura nuclear, e índices de colocalização mais altos para duas sondas de repetições adjacentes das extremidades cromossômicas, Rep20 e telômeros. Em resumo, nossos resultados mostram que o tipo de protocolo de fixação utilizado antes da realização do desenvolvimento de FISH é um fator crucial para a conservação apropriada da arquitetura nuclear.Growing evidence points that transcription spatial organization is an important epigenetic factor for eukaryotes gene regulation. In mammalian cells, genes are transcribed in discrete nuclear structures known as transcription factories, and developmentally co-regulated, functionally related genes have been shown to share factories. Plasmodium falciparum shows a remarkably complex pattern of gene expression during the erythrocytic cycle, paradoxically contrasting with the low number of putative transcription factors encoded by its genome. However, epigenetic mechanisms are important for gene regulation in this parasite. In this thesis, we investigated transcription organization of P. falciparum in order to determine if nuclear architecture is related with developmentally regulated gene expression. To this aim, we have labeled nascent transcrips with BrUTP in P. falciparum erythrocytic forms. Transcription in organized in discrete nuceloplasmic foci, the transcription factories. Automated 3D analysis of confocal images shows the number and intensity of transcription factories change during the erythrocytic cycle, correlating with the number of genes expressed at each stage but not with the nuclear volume. The low number of factories indicates that active genes share factories while being transcribed. Unexpectedly, factories spatially redistribute from ring to trophozoites, being the nuclear periphery the preferential transcription zone in rings while in trophozoites factories are equally distributed throughout the nucleoplasm. We also observed that RNApolymerase I transcription occurs mostly at central nuclear areas in trophozoites, suggesting that nucleolar components may be dispersed upon S phase transition. Like in higher eukaryotes, in P. falciparum transcription factories occur on nuclear areas with low chromatin density. Immunofluorescence analysis of P. falciparum nuclear markers combined with BrUTP incorporation show that transcription factories are a novel and exclusive nuclear compartment, different from the silent compartment defined by PfSir2A or the active chromatin compartment defined by H4ac and H3K79me3. In order to study the spatial organization of chromatin, and how co-regulated, functionally related genes interact with transcription factories, we decided to perform fluorescence in situ hybridization (FISH). While performing FISH with published protocols, we observed great variation in the parasite nuclear morphology, prompting us to optimize this technique. Using automated image analysis, we show that parasites dehydrated by air-drying and fixed for short periods at room temperature present after FISH higher intra-population variation of nuclear shape and volume, as well as almost two-times higher relative volume values, than parasite nuclei fixed in suspension for long periods at low temperatures. We also observe that longer fixation in suspension leads to improved conservation of the nuclear structure, with chromosome end clusters preferentially locating at the nuclear periphery, and higher colocalization indexes for two adjacent chromosome end probes, Rep20 and telomere. Overall, our results show that the type of fixation protocol applied prior to FISH is crucial for the conservation of nuclear architecture.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Ministério da Educação, Ciência e Tecnologia da Coreia do Sul (MEST)Governo da província de Gyeonggi da Coreia do SulInstituto Coreano de Informação Científica e Tecnológica (KISTI)BV UNIFESP: Teses e dissertaçõe