The local microenvironment of hematopoietic stem cells (HSCs) in the bone marrow -referred to as stem cell niche- is thought to regulate the balance of stem cell maintenance and differentiation by a complex interplay of extrinsic signals including spatial constraints, extracellular matrix (ECM) components and cell-cell interactions. To dissect the role of niche ECM components, a set of well-defined matrix biomolecular coatings including fibronectin, laminin, collagen IV, tropocollagen I, heparin, heparan sulphate, hyaluronic acid and co-fibrils of collagen I with heparin or hyaluronic acid were prepared and analyzed with respect to adhesive interactions of human CD133+ HSCs in vitro. ECM molecule dependent adhesion areas as well as fractions of adherent HSCs were assessed by reflection interference contrast microscopy and differential interference contrast microscopy. HSCs, so far mostly classified as suspension cells, exhibited intense adhesive interactions with fibronectin, laminin, collagen IV, heparin, heparan sulphate, and collagen I based co-fibrils. An integrin mediated adhesion on fibronectin and a L-selectin mediated adhesion on heparin pointed to specific interactions based on different adhesion mechanisms. As a consequence of HSC adhesion to molecules of the vascular and the endosteal regions, both regions were confirmed as possible stem cell niches and adhesive signals were suggested as potential regulators of stem cell fate. Furthermore, the impact of a spatially organized ECM on the HSC behavior was analyzed by single cell tracking. These studies required the development of engineered three-dimensional, ECM coated microcavities with the option for single cell tracking. A semi-automated cell-tracking tool was established to accelerate data access from time-lapse image sequences. From this analysis it was possible to reveal the genealogy, localization, morphology and migration of single HSCs over a time period of 4 days. A decreased cycling frequency was observed depending on the HSC localization in the spatially constraining microcavities. Besides the revealed impact of spatial constraints on HSC fate, the newly engineered ECM-coated microcavity setup and the semi-automated cell tracking tool provide new options to study the cell fate in engineered microenvironments at single cell level for other cell types ex vivo. / Die lokale Mikroumgebung von Blutstammzellen (BSZ) im Knochenmark, bezeichnet als Stammzellnische, reguliert das Gleichgewicht von Stammzellerhaltung und -differenzierung durch ein komplexes Zusammenspiel von extrinsischen Signalen wie räumliche Beschränkungen, Komponenten der extrazellulären Matrix (EZM) und Zell-Zell Wechselwirkungen. Um die Rolle der EZM-Komponenten zu analysieren, wurden definierte Beschichtungen von Fibronektin, Laminin, Kollagen IV, monomerem Kollagen I, Heparin, Heparan Sulphat, Hyaluronsäure und Co-Fibrillen aus Kollagen I und Heparin oder Hyaluronsäure hergestellt und in vitro bezüglich der adhäsiven Wechselwirkungen von humanen CD133+ BSZ untersucht. Die Adhäsionsflächen und der Anteil adhärenter Zellen wurden in Abhängigkeit von der EZM-Beschichtung mittels Reflexions- Interferenz-Kontrast-Mikroskopie und Differentieller Interferenz Kontrast Mikroskopie bestimmt. BSZ, bisher als Suspensionszellen definiert, zeigten intensive adhäsive Wechselwirkungen mit Fibronektin, Laminin, Kollagen IV, Heparin, Heparan Sulphat und den Co-Fibrillen. Eine Integrin abhängige Adhäsion auf Fibronektin und eine L-Selektin abhängige Adhäsion auf Heparin, wiesen auf spezifische Wechselwirkungen hin, die auf unterschiedlichen Mechanismen basieren. Aufgrund der Adhäsion von BSZ sowohl zu Molekülen der vaskulären als auch der endostealen Knochenmarkregion, wurden beide Bereiche als mögliche Stammzellnische bestätigt. Adhäsive Signale sind potentielle Regulatoren der Stammzellentwicklung. Im Weiteren wurde der Einfluss einer räumlich beschränkenden EZM auf das Verhalten der BSZ durch Einzelzellverfolgung untersucht. Diese Studien erforderten die Entwicklung von dreidimensionalen EZM-beschichteten Mikrokavitäten, die das Verfolgen einzelner Zellen ermöglichten. Es wurde ein halbautomatischer Algorithmus für die Zellverfolgung etabliert, um die Datengenerierung von den Zeitreihenaufnahmen zu beschleunigen. Die Analysen ermöglichten Aussagen über die Genealogie, Lokalisierung, Morphologie und Migration einzelner BSZ während einer Analysenzeit von 4 Tagen. Eine verringerte Zellteilungsaktivität wurde in Abhängigkeit von der BSZ Lokalisierung innerhalb der räumlich einschränkenden Mikrokavitäten festgestellt. Neben diesen Erkenntnissen bieten die entwickelten Mikrokavitäten und die etablierte Einzelzellverfolgung neue Möglichkeiten auch andere Zelltypen auf Einzelzellniveau ex vivo zu untersuchen.

Mesenchymale Stammzellen (MSC) werden bereits in klinischen Studien zur Behandlung verschiedener Krankheiten eingesetzt. Über deren Wirkmechanismus und Verbleib nach Applikation ist jedoch noch wenig bekannt. Die in vivo-Nachverfolgung markierter MSC mittels Magnetresonanztomographie stellt eine mögliche Methode zur Erlangung weiterer Erkenntnisse dar. Zu diesem Zweck können die MSC mittels superparamagnetischen Eisenoxid (SPIO)-Partikeln markiert werden. In dieser Arbeit wurden 3 verschiedene SPIO-Produkte zur Markierung oviner und equiner MSC verwendet: Endorem™, Resovist® und Molday ION Rhodamine B™. Die Produkte wurden hinsichtlich ihrer Einflüsse auf die biologischen Eigenschaften der MSC, ihrer Markierungseffizienz und –selektivität verglichen. Desweiteren wurde die produktspezifische magnetresonanztomographische Nachverfolgbarkeit der SPIO-markierten MSC untersucht. Weiterführendes Ziel war die Selektion des am besten geeigneten SPIO-Produktes für die Verwendung in einem in vivo-Großtierversuch zur magnetresonanztomographischen Nachverfolgung SPIO-markierter MSC nach Applikation in arthrotische Gelenke. Die MSC wurden dazu aus dem Knochenmark von je 5 gesunden Schafen und Pferden isoliert, bis zur Passage 4 (P4) expandiert und schließlich mit den verschiedenen SPIO-Produkten markiert. Unmarkierte MSC der gleichen Tiere dienten zur Kontrolle. Proliferationsvermögen sowie tripotentes Differenzierungspotential wurden in vitro untersucht. Zur Evaluierung von Markierungsselektivität und -effizienz der SPIO-Produkte wurden die MSC ab der P4 bis zur P7 wöchentlich passagiert. Ein semiquantitatives histologisches Auswertungssystem basierend auf der Preußisch Blau-Färbung sowie T2*w-GRE-Sequenzen an einem 0,5T-MRT-System wurden zur Evaluierung genutzt. Markierungsselektivität bezeichnete die intra- oder extrazelluläre Lokalisation der SPIO-Partikel. Markierungseffizienz beschrieb die Menge intrazellulär vorhandener SPIO-Partikel. Es wurde gezeigt, dass sich ovine und equine MSC mit allen 3 untersuchten SPIO-Produkten erfolgreich markieren ließen. Die Ergebnisse der in vitro-Untersuchungen ergaben keine Unterschiede zwischen SPIO-markierten und unmarkierten MSC hinsichtlich des Proliferationsvermögens, der adipogenen oder osteogenen Differenzierungsfähigkeit. Jedoch wurde eine deutliche Verminderung des chondrogenen Differenzierungspotentials SPIO-markierter MSC beobachtet, welche von der Menge intrazellulär vorhandener SPIO-Partikel und somit von der Markierungseffizienz abhängig war. Zum Zeitpunkt der initialen Markierung konnte nur Molday ION Rhodamine B™ eine selektive und effiziente Zellmarkierung gewährleisten. Mit Endorem™ konnte eine selektive, jedoch keine ausreichend effiziente Zellmarkierung erreicht werden. Resovist® dagegen bewirkte zwar eine effiziente, aber sehr unselektive initiale Zellmarkierung: Mittels Preußisch Blau-Färbung wurde gezeigt, dass große Mengen von SPIO-Partikeln nur extrazellulär anhefteten. Die 3 verschiedenen SPIO-Produkte führten weiterhin zu unterschiedlich starken hypointensen MRT-Signalen der markierten MSC, welche im Verlauf der 3-wöchigen Versuchsdauer bei allen 3 Produkten stetig abnahmen. Unmarkierte MSC waren isointens, also mittels MRT nicht darstellbar und daher nicht nachverfolgbar. Stets verursachten Resovist®-markierte MSC das stärkste hypointense MRT-Signal, gefolgt von Molday ION Rhodamine B™ und Endorem™. Resovist®-markierte MSC konnten mittels MRT bei beiden Spezies über den längsten Zeitraum nachverfolgt werden (ovine MSC bis 16 Tage, equine MSC bis 23 Tage nach Markierung). Aufgrund der exzellenten initialen Markierungseigenschaften (hohe Markierungsselektivität und –effizienz sowie gute Nachverfolgbarkeit) eignet sich Molday ION Rhodamine B™ besonders gut für die SPIO-Markierung von MSC zur Nachverfolgung mittels MRT. Molday ION Rhodamine B™ verspricht somit eine erfolgreiche Anwendung in einem in vivo-Versuch zur magnetresonsztomographischen Nachverfolgung von MSC nach Applikation in arthrotische Gelenke. / Mesenchymal stem cells (MSC) are already used in clinical studies for treatment of different diseases. However, their mechanism of action and fate after application are still not fully understood. In vivo tracking of labeled MSC via magnetic resonance imaging (MRI) is a possible method to achive further knowledge. For this purpose MSC can be labelled with superparamagnetic iron oxide (SPIO) particles. For this study 3 different SPIO products were employed for labelling of ovine and equine MSC: Endorem™, Resovist®,, and Molday ION Rhodamine B™. The products were compared in terms of their influence on biologic behaviour of the MSC, their labelling efficiency, and selectivity. Furthermore, product specific magnetic resonance traceability of SPIO labelled MSC was evaluated. Final aim was the selection of the most suitable SPIO product to be used in an in vivo large animal study employing MRI tracking of SPIO labelled MSC after application into osteoarthritic joints. MSC therefore, were isolated from bone marrow of each 5 healthy sheep and horses, expanded up to passage 4 (p4), and labelled by the different SPIO products. Unlabelled MSC from the same animals served as control. Proliferation potential and tripotent differentiation capacities were assessed in vitro. For evaluation of labelling selectivity and efficiency of the SPIO products MSC were passaged weekly from p4 up to p7. Semiquantitative histological scoring based on Prussian blue staining and images using T2*w GRE sequences in a 0.5T MRI system were used. Labelling selectivity describes the intra- or extracellular localisation of the SPIO particles. Labelling efficiency describes the amount of intracellular SPIO particles. It was shown that ovine and equine MSC could be successfully labelled by all 3 evaluated SPIO products. The results of the in vitro experiments did not show differences between labelled and unlabelled MSC in terms of proliferation potential, adipogenic or osteogenic differentiation capacities. However, an inhibited chondrogenic differentiation capacity of SPIO labelled MSC was observed, which was dependend on the amount of intracellular SPIO particles and therefore, also on labelling efficiency. At the time of initial labelling, only Molday ION Rhodamine B™ showed selective and efficient cell labelling. With Endorem™ selective, but not efficient cell labelling was achieved. Resovist®, in contrast, caused efficient but very unselective initial cell labelling: By Prussian blue staining it was shown that large amounts of SPIO particles were attached extracellularly. These 3 different SPIO products led to variable hypointense MRI signals of the labelled MSC which decreased in all 3 products during the 3 week study period. Unlabelled MSC were isointense, thus not visible, and therefore, not traceable using MRI. At every point of time, Resovist® labelled MSC resulted in the most hypointense MR signals, followed by Molday ION Rhodamine B™ and Endorem™. Resovist® labelled MSC were traced over the longest time span (ovine MSC until 16 days, equine MSC until 23 days post labelling). Due to excellent initial labelling properties (high labelling efficiency and selectivity, good traceability) Molday ION Rhodamine B™ suits best for SPIO labelling of MSC to be tracked by MRI. Molday ION Rhodamine B™ therefore, promises a successful use in an in vivo study using MRI for MSC tracking after application into osteoarthritic joints.

No description available.

The two defining features of mouse embryonic stem (ES) cells are self-renewal and naive pluripotency, the ability to give rise to all cell lineages in the adult body. In addition to being a unique and interesting cell type, pluripotent ES cells have demonstrated their potential for continued advancements in biomedical science. Currently, there is an improved understanding in the chemical signals and the gene regulatory network responsible for the maintenance of ES cells in the naive pluripotent state. However, less is understood about how ES cells exit pluripotency. My main aim is to study the dynamics and the factors affecting the irreversible exit from pluripotency. Expression of the reporter Rex1-GFPd2, which is inactivated upon exit from naive pluripotency, was analyzed by quantitative long-term single-cell imaging over many generations. This technique allowed chemical, physical, and genealogical information to be recorded during the transition to exit. Culture conditions that provided homogeneous populations were used in all assays and these data were validated against bulk-culture data where appropriate. Changes in real-time cell behavior were seen in cell-cell contact, motility, and cell-cycle duration. Undifferentiated ES cells form tightly joined colonies, with cells that exhibit low motility and a constant cell-cycle duration. Exit is associated with increasing cell motility, decreased cell-cell contact, and an acceleration in cell proliferation. The onset of exit is associated with a sudden and irreversible inactivation of the Rex1-GFPd2 reporter. This inactivation is asynchronous, as it occurs at different times and in different generations during ES cell differentiation. However, examination of daughter cells generated from the same mother revealed a high level of synchronicity. Further investigation revealed that high levels of correlation in cell-cycle duration and Rex1-GFPd2 expression exist between differentiating sister and cousin cells, providing strong evidence that cell potency is inherited symmetrically in cell divisions during exit $\textit{in vitro}$. How cells change fate is a fundamental question in developmental biology. Knowing the cellular dynamics during the transition out of naive pluripotency is important for harnessing the potential of ES cells and understanding how cell fate decisions are made during embryonic development. The quantification of the timing of exit from naive pluripotency coupled with identifiable changes in cellular behaviors, such as motility, cell size, and cell-cycle duration, enhances the understanding of how cell fate changes are regulated during directed differentiation.

Cardiac tissue engineering promises to create therapeutic tissue replacements for repair of diseased native myocardium. The main goals of this thesis were four-fold: 1) to evaluate cardiac tissues engineered using multiple cell types including endothelial cells (EC), fibroblasts (FB), and cardiomyocytes (CM); 2) to spatiotemporally track cells in organoids and optimize their seeding percentages for improved function; 3) to enhance vascular cord formation through sequential versus simultaneous seeding of ECs and FBs; and 4) to perform mechanistic studies to elucidate the role of soluble factors in cell-cell communication. Microscale templates fabricated from photocrosslinkable poly(ethylene glycol) diacrylate (PEG-DA) were used for all studies for rapid screening. When ECs and FBs were precultured for two days prior to seeding enriched CMs, cells self-assembled into three-dimensional, beating organoids, compared to simultaneously tricultured EC/ FB / CM which formed non-contractile clusters. Fluorescent dyes were used to label and track each cell type for up to 4 days, demonstrating an even distribution of cells within precultured organoids versus EC clustering in simultaneous triculture. When ECs were seeded first, followed by FBs 24 hours later and CMs 48 hours later, vascular-like cords formed that persisted with time in a seeding density-dependent manner. Vascular endothelial growth factor (VEGF) signaling was quantified, showing higher endogenous VEGF secretion rates in sequential preculture (16.6 ng/mL/hr) compared to undetectable VEGF secretion in simultaneous triculture. Blocking of endogenous VEGF signaling through addition of VEGF antibody / VEGFR2 inhibitor resulted in a significant decrease in mRNA and protein expression of the key cardiac gap junctional marker connexin-43. These findings provide a foundation for future work into the mechanisms governing functional cardiac tissue engineering performance and may aid in the development of novel therapies for heart failure based on growth factor signaling and engineering of vascularized, clinically relevant cardiac tissue patches.

Cardiac tissue engineering promises to create therapeutic tissue replacements for repair of diseased native myocardium. The main goals of this thesis were four-fold: 1) to evaluate cardiac tissues engineered using multiple cell types including endothelial cells (EC), fibroblasts (FB), and cardiomyocytes (CM); 2) to spatiotemporally track cells in organoids and optimize their seeding percentages for improved function; 3) to enhance vascular cord formation through sequential versus simultaneous seeding of ECs and FBs; and 4) to perform mechanistic studies to elucidate the role of soluble factors in cell-cell communication. Microscale templates fabricated from photocrosslinkable poly(ethylene glycol) diacrylate (PEG-DA) were used for all studies for rapid screening. When ECs and FBs were precultured for two days prior to seeding enriched CMs, cells self-assembled into three-dimensional, beating organoids, compared to simultaneously tricultured EC/ FB / CM which formed non-contractile clusters. Fluorescent dyes were used to label and track each cell type for up to 4 days, demonstrating an even distribution of cells within precultured organoids versus EC clustering in simultaneous triculture. When ECs were seeded first, followed by FBs 24 hours later and CMs 48 hours later, vascular-like cords formed that persisted with time in a seeding density-dependent manner. Vascular endothelial growth factor (VEGF) signaling was quantified, showing higher endogenous VEGF secretion rates in sequential preculture (16.6 ng/mL/hr) compared to undetectable VEGF secretion in simultaneous triculture. Blocking of endogenous VEGF signaling through addition of VEGF antibody / VEGFR2 inhibitor resulted in a significant decrease in mRNA and protein expression of the key cardiac gap junctional marker connexin-43. These findings provide a foundation for future work into the mechanisms governing functional cardiac tissue engineering performance and may aid in the development of novel therapies for heart failure based on growth factor signaling and engineering of vascularized, clinically relevant cardiac tissue patches.

Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques. In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level. To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs. The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. / <p>QC 20130927</p>

This dissertation deals with customised image analysis tools in cancer research. In the field of biomedical sciences, mathematical imaging has become crucial in order to account for advancements in technical equipment and data storage by sound mathematical methods that can process and analyse imaging data in an automated way. This thesis contributes to the development of such mathematically sound imaging models in four ways: (i) automated cell segmentation and tracking. In cancer drug development, time-lapse light microscopy experiments are conducted for performance validation. The aim is to monitor behaviour of cells in cultures that have previously been treated with chemotherapy drugs, since atypical duration and outcome of mitosis, the process of cell division, can be an indicator of successfully working drugs. As an imaging modality we focus on phase contrast microscopy, hence avoiding phototoxicity and influence on cell behaviour. As a drawback, the common halo- and shade-off effect impede image analysis. We present a novel workflow uniting both automated mitotic cell detection with the Hough transform and subsequent cell tracking by a tailor-made level-set method in order to obtain statistics on length of mitosis and cell fates. The proposed image analysis pipeline is deployed in a MATLAB software package called MitosisAnalyser. For the detection of mitotic cells we use the circular Hough transform. This concept is investigated further in the framework of image regularisation in the general context of imaging inverse problems, in which circular objects should be enhanced, (ii) exploiting sparsity of first-order derivatives in combination with the linear circular Hough transform operation. Furthermore, (iii) we present a new unified higher-order derivative-type regularisation functional enforcing sparsity of a vector field related to an image to be reconstructed using curl, divergence and shear operators. The model is able to interpolate between well-known regularisers such as total generalised variation and infimal convolution total variation. Finally, (iv) we demonstrate how we can learn sparsity promoting parametrised regularisers via quotient minimisation, which can be motivated by generalised Eigenproblems. Learning approaches have recently become very popular in the field of inverse problems. However, the majority aims at fitting models to favourable training data, whereas we incorporate knowledge about both fit and misfit data. We present results resembling behaviour of well-established derivative-based sparse regularisers, introduce novel families of non-derivative-based regularisers and extend this framework to classification problems.

The goal of this thesis is the development of a tracking algorithm for populations of unmarked cancer cells that migrate in 3D in vitro gels. The tracking algorithm is intended to be a tool for analysing the motility of large population (i.e. hundreds) of cells in the context of the anti-migratory drug development and more specifically drug screening. In oncology, cancer cell migration plays pivotal roles in the spread of cancer cells from a primary tumor site to neighboring and secondary sites, i.e. the processes of tissue invasion and metastasis. Preventing such processes represents an important therapeutic approach to cancer treatment. Providing tools able to test potential anti-migratory drugs thus constitutes currently a real need in oncology therapy. The goal of drug screening in this context aims to rapidly and efficiently test the anti-migratory effects of many experimental conditions on cancer cell populations.<p>The focus in this thesis lies in two specific aspects that are important in anti-migratory drug screening: tracking cells inside an in vitro 3D environment and doing so using unmarked cells. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished

Par leur stabilité, leur petite taille et leur nature monomérique, les domaines variables des immunoglobulines à chaînes lourdes, ou Nanobodies (Nb), sont incontournables en diagnostic et recherche médicale. Pourtant, leur utilisation en agro-biotechnologies demeure confidentielle.Dans l'idée de les utiliser pour étudier et combattre le Grapevine fanleaf virus (GFLV), responsable de la maladie du court-noué très préjudiciable à l'économie viticole mondiale, j'ai produit une collection de Nb spécifiques du GFLV.Fusionné à une protéine fluorescente et exprimé en plante de façon stable, un de ces Nb (alors appelé Chromobody, Cb) a conféré une haute résistance au GFLV inoculé mécaniquement ou transmis par nématodes.Le potentiel du Cb comme biocapteur a été validé par le suivi in vivo d’un isolat contournant la résistance mais toujours reconnu par le Cb. La structure du complexe Nb/GFLV a été résolue à 2,8 Å et révèle la zone occupée par le Nb à la surface de la capside.Ces résultats ouvrent des perspectives innovantes pour la compréhension du cycle infectieux d'un phytovirus et l'élaboration de nouvelles stratégies de lutte antivirale. / Due to their small size, high stability and strict monomeric nature, Nanobodies (Nbs) deriving from camelids heavy chain only antibodies have proven very valuable as diagnostic and therapeutic tools. However their use in agro biotechnology remains limited.In order to apply Nbs to the study and the control of grapevine fanleaf degeneration, I produced acollection of Nbs against Grapevine fanleaf virus (GFLV), the causal agent of this devastating disease worldwide.When fused to a fluorescent protein and stably expressed in plants, one of these Nbs (calledChromobody, Cb) conferred high resistance to GFLV, whether inoculated mechanically or by vector-mediated transmission.The identification of an isolate overcoming the resistance but still bound by the Cb allowed real-time tracking of the infection showing the high potential of Cbs as biosensors.The cryoEM structure of the Nb/GFLV complex was obtained at 2,8 Å and provides a clear picture of the footprint of the Nb on the surface of the GFLV capsid.These results pave the way for the innovative use of Nbs to unravel viral life cycle and to counter viral diseases.