Quantitative automated analysis of host-pathogen interactions

This work aims to broaden knowledge about neutrophil biology in their interaction with fungi species that most frequently cause invasive fungal diseases (IFD). The questions addressed include the alteration of neutrophil morphology after interaction with Candida albicans or C. glabrata, revealing factors that modulate the production and composition of neutrophil-derived extracellular vesicles (EVs) obtained in confrontation assay with conidia of Aspergillus fumigatus and analysing EVs activity against this fungus. Alongside fundamental interests, those questions have important applied aspects in the medicine of IFD. In particular, for diagnostic purposes and infection process monitoring. The results of this work include: 1 a novel segmentation and tracking algorithm which is capable of working with low-contrast cell images, producing accurate cell contours and providing data about positions of clusters, which would improve further analysis; 2 a novel workflow algorithm for analysis of neutrophil continuous morphological spectrum without consensus-based manual annotation; 3 quantitative evidence that morphodynamics of isolated neutrophils depends on the infectious agent (C. albicans or C. glabrata) used in whole blood infection assay; 4 quantitative evidence that neutrophil-derived extracellular vesicles, obtained in confrontation assays with conidia of A. fumigatus could inhibit hyphae development and damage hyphae cell wall; 5 quantitative evidence that EVs inhibition activity is strain-specific

NANOSCALE INVESTIGATION OF NUCLEAR STRUCTURES BY TIME-RESOLVED MICROSCOPY

The eukaryotic cell nucleus is composed by heterogeneous biological structures, such as the nuclear envelope (NE) and chromatin. At a morphological level, chromatin organization and its interactions with nuclear structures, such as nuclear lamina (NL) and nuclear pore complex (NPC), are suggested to play an essential role in the regulation of gene activity, which involves the packaging of the genome into transcriptionally active and inactive sites, bound to healthy cell proliferation and maintenance. However, the processes governing the relation between nuclear structures and gene regulation are still unclear. For this reason, the advanced microscopy methods represent a powerful tool for imaging nuclear structures at the nanometer level, which is essential to understand the effect of nuclear interactions on genome function. The nanometer information may be achieved either through the advanced imaging techniques in combination with fluorescence spectroscopy or with the help of super-resolution methods, increasing the spatial resolution of the conventional optical microscopy. In this thesis, I implemented a double strategy based on a novel FLIM-FRET assay and super resolution SPLIT-STED method for the investigation of the chromatin organization and nuclear envelope components (lamins and NPC) at the nanoscale, in combination with the phasor analysis. The phasor approach can be applied to several fluorescence microscopy techniques abled to provide an image with an additional information in a third channel. Phasor plot is a graphical representation, which decodes the fluorescence dynamics encoded in the image, revealing a powerful tool for the data analysis in time-resolved imaging. The Chapter 1 of the thesis is characterized by an Introduction, which provides an overview on the chromatin organization at the nanoscale and the description of the several advanced fluorescence microscopy techniques used for its investigation. They are broadly divided into two main categories: the advanced imaging techniques, such as Fluorescence Correlation Spectroscopy (FCS), single particle tracking (SPT) and Fluorescence Recovery After Photobleaching (FRAP), Forster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) and the super-resolution techniques, which include Stimulated Emission Depletion (STED), Structured Illumination Microscopy (SIM) and single molecule localization microscopy (SMLM). Following, Chapter 2 focus on the capabilities of the phasor approach in time-resolved microscopy, as a powerful tool for the analysis of the experimental data. After a description of the principles of time-domain and frequency-domain measurements, in this section are explained the rules of the phasor analysis and its applications in different fluorescence microscopy techniques. In Chapter 3, I present a FRET assay, based on the staining of the nuclei with two DNA-binding dyes (e.g. Hoechst 33342 and Syto Green 13) by using frequency-domain detection of FLIM and the phasor analysis in live interphase nuclei. I show that the FRET level strongly depends on the relative concentration of the two fluorophores. I describe a method to correct the values of FRET efficiency and demonstrate that, with this correction, the FLIM-FRET assay can be used to quantify variations of nanoscale chromatin compaction in live cells. In Chapter 4, the phasor analysis is employed to the improvement of the resolving power of the super-resolution STED microscopy. I describe a novel method to investigate nuclear structures at the nanometer level, known as SPLIT (Separation of Photons by Lifetime Tuning), developed by my group in last years. By using the phasor approach, the SPLIT technique decodes the variations of spectroscopic parameters of fluorophores, such as lifetime and fluorescence intensity, due to the effect of the modulated depletion power of the STED technique, increasing the resolving power. In this chapter, I develop the concept of the SPLIT method modulating the excitation pattern during the image acquisition to overcome its limitation linked to the photobleaching effect and the signal-to-noise ratio

New algorithms for the analysis of live-cell images acquired in phase contrast microscopy

La détection et la caractérisation automatisée des cellules constituent un enjeu important dans de nombreux domaines de recherche tels que la cicatrisation, le développement de l'embryon et des cellules souches, l’immunologie, l’oncologie, l'ingénierie tissulaire et la découverte de nouveaux médicaments. Étudier le comportement cellulaire in vitro par imagerie des cellules vivantes et par le criblage à haut débit implique des milliers d'images et de vastes quantités de données. Des outils d'analyse automatisés reposant sur la vision numérique et les méthodes non-intrusives telles que la microscopie à contraste de phase (PCM) sont nécessaires. Comme les images PCM sont difficiles à analyser en raison du halo lumineux entourant les cellules et de la difficulté à distinguer les cellules individuelles, le but de ce projet était de développer des algorithmes de traitement d'image PCM dans Matlab® afin d’en tirer de l’information reliée à la morphologie cellulaire de manière automatisée. Pour développer ces algorithmes, des séries d’images de myoblastes acquises en PCM ont été générées, en faisant croître les cellules dans un milieu avec sérum bovin (SSM) ou dans un milieu sans sérum (SFM) sur plusieurs passages. La surface recouverte par les cellules a été estimée en utilisant un filtre de plage de valeurs, un seuil et une taille minimale de coupe afin d'examiner la cinétique de croissance cellulaire. Les résultats ont montré que les cellules avaient des taux de croissance similaires pour les deux milieux de culture, mais que celui-ci diminue de façon linéaire avec le nombre de passages. La méthode de transformée par ondelette continue combinée à l’analyse d'image multivariée (UWT-MIA) a été élaborée afin d’estimer la distribution de caractéristiques morphologiques des cellules (axe majeur, axe mineur, orientation et rondeur). Une analyse multivariée réalisée sur l’ensemble de la base de données (environ 1 million d’images PCM) a montré d'une manière quantitative que les myoblastes cultivés dans le milieu SFM étaient plus allongés et plus petits que ceux cultivés dans le milieu SSM. Les algorithmes développés grâce à ce projet pourraient être utilisés sur d'autres phénotypes cellulaires pour des applications de criblage à haut débit et de contrôle de cultures cellulaires.Automated cell detection and characterization is important in many research fields such as wound healing, embryo development, immune system studies, cancer research, parasite spreading, tissue engineering, stem cell research and drug research and testing. Studying in vitro cellular behavior via live-cell imaging and high-throughput screening involves thousands of images and vast amounts of data, and automated analysis tools relying on machine vision methods and non-intrusive methods such as phase contrast microscopy (PCM) are a necessity. However, there are still some challenges to overcome, since PCM images are difficult to analyze because of the bright halo surrounding the cells and blurry cell-cell boundaries when they are touching. The goal of this project was to develop image processing algorithms to analyze PCM images in an automated fashion, capable of processing large datasets of images to extract information related to cellular viability and morphology. To develop these algorithms, a large dataset of myoblasts images acquired in live-cell imaging (in PCM) was created, growing the cells in either a serum-supplemented (SSM) or a serum-free (SFM) medium over several passages. As a result, algorithms capable of computing the cell-covered surface and cellular morphological features were programmed in Matlab®. The cell-covered surface was estimated using a range filter, a threshold and a minimum cut size in order to look at the cellular growth kinetics. Results showed that the cells were growing at similar paces for both media, but their growth rate was decreasing linearly with passage number. The undecimated wavelet transform multivariate image analysis (UWT-MIA) method was developed, and was used to estimate cellular morphological features distributions (major axis, minor axis, orientation and roundness distributions) on a very large PCM image dataset using the Gabor continuous wavelet transform. Multivariate data analysis performed on the whole database (around 1 million PCM images) showed in a quantitative manner that myoblasts grown in SFM were more elongated and smaller than cells grown in SSM. The algorithms developed through this project could be used in the future on other cellular phenotypes for high-throughput screening and cell culture control applications

Localisation Super-resolution Imaging Using Germanium Quantum Dots.

PhDNovel fluorescent quantum dots of small size, tunable light emission wavelength and high compatibility with biological systems are of great significance to light microscopy super-resolution imaging. In this thesis, colloidal germanium quantum dots of 3.8 nm size have been investigated as a novel fluorescent probe for cell imaging. Two single molecule localisation super-resolution methods were explored: one utilised QDs blinking statistics and the other one was based on intrinsic QDs size dispersion. We found that the blinking super-resolution strategy which combined the usage of blinking QDs and spinning disk confocal imaging has led to less than seven minutes collection time for 2000 image frames. High precision temporal separation of single molecules has been achieved on Ge QDs and CdSe QDs labelled fixed Hela cell. The spectroscopic super-resolution strategy that combined the usage of size dependent light emission QDs and spectra imaging, resulted in a 1.6 seconds data acquisition time. Spectroscopic separation and high precision single molecule localisation has been demonstrated using Ge QDs and CdSe QDs labelled fixed Hela cell samples. We compared various localisation algorithms when applied to the two superresolution methods we studied. We found that they did not work well with our data. Consequently, we developed two MATLAB-based localisation algorithms. The first algorithm used the independent component analysis (ICA) model to analyse the blinking stochastic imaging data, whilst the other used the Gaussian mixed model (GMM) to analyse the spectroscopic separation imaging data. 6 Contents We also conducted comparative toxicity tests of these novel Ge QDs with a typical of-the-shelf system. The cell toxicity of Ge QDs was found to be less than that of CdSe/ZnS QDs. For instance, 25 nM Ge QDs in 1 mL Hela cell solution did not cause observable cells apoptosis in 24 hours. It caused 15% cells apoptosis after 3 days, rather than 35% for CdSe QDs at the same concentration. In addition, long term live cell imaging with QDs revealed that Ge QDs had not significantly changed cellular morphology within a 90 hour period.China Scholarship Council and Queen Mary, University of Londo

Real-time blood oxygenation tomography with multispectral photoacoustics

Multispectral photoacoustics is an emerging biomedical imaging modality which combines the penetration depth and resolution of high frequency medical ultrasonography with an optical absorption contrast. This enables tomographic imaging of blood oxygen saturation, a functional biomarker with wide applications. Already, photoacoustic imaging (PAI) is widely applied for small animal imaging in preclinical research. While PAI is a multiscale modality, its translation to clinical research and interventional use remains challenging. The objective of this thesis was to investigate the usefulness of multispectral PAI as a technique for interventional tomographic imaging of blood oxygenation. This thesis presents open challenges alongside research contributions to address them. These contributions are, (1) The design and implementation of an interventional PAI system, (2) Methods for real-time photoacoustic (PA) image processing and quantification of tissue absorption and blood oxygenation, and finally (3) the application of multispectral PAI to translational neurosurgical research – performing the first high spatiotemporal resolution tomography of spreading depolarization, and at the same time the first interventional PAI on any gyrencephalic (folded) brain. Such interventional imaging in neurology is one of many promising fields of application for PAI

Microscopy and Analysis

Microscopes represent tools of the utmost importance for a wide range of disciplines. Without them, it would have been impossible to stand where we stand today in terms of understanding the structure and functions of organelles and cells, tissue composition and metabolism, or the causes behind various pathologies and their progression. Our knowledge on basic and advanced materials is also intimately intertwined to the realm of microscopy, and progress in key fields of micro- and nanotechnologies critically depends on high-resolution imaging systems. This volume includes a series of chapters that address highly significant scientific subjects from diverse areas of microscopy and analysis. Authoritative voices in their fields present in this volume their work or review recent trends, concepts, and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area

Development and validation of kinase activity reporters for the dynamic study of cell response modalities by microscopy

Necroptosis is defined as a caspase-independent programmed cell death and relies on a signaling pathway involving two serine-threonine kinases: Receptor-Interacting Protein Kinase 1 and 3 (RIPK1 and RIPK3) and the pseudo-kinase Mixed-Lineage Kinase Like (MLKL). Activation of Extracellular signal-Regulated Kinases 1 and 2 (ERK1/2) was reported to be involved in different modes of programmed cell death. It is now accepted that the regulation of the duration, magnitude and subcellular compartmentalization of ERK1/2 activity by specific spatio-temporal regulators is interpreted by the cell towards cell fate determination. ERK1/2 inhibition delays TNFα-induced necroptosis in L929 cells in a dose dependent manner but did not block it, providing arguments for a pro-necrotic function of ERK1/2. In this context, a compartmentalized biphasic phosphorylation of ERK1/2 was observed. Our results indicate a RIPK1-dependent phosphorylation of ERK1/2. Owing to the importance of ERK1/2 spatio-temporal dynamics in determining cellular responses, we developed a new reporter of ERK2 localization named ERK2-LOC. We observed a transient translocation of ERK2 when necroptosis was triggered in L929 upon TNFα stimulation, followed by progressive ERK2 accumulation in the nucleus. ERK1/2 activities were monitored during necroptosis using a FRET-based kinase biosensor for ERK1/2 (ERK1/2-ACT). Using ERK1/2-ACT, a dedicated spatio-temporal signature of ERK1/2 activity was recorded during necroptosis. Finally, to correlate ERK1/2 activity code with necroptosis occurrence, we also engineered a first generation of FRET biosensors to report on both RIPK1 and RIPK3 activities during necroptosis

Development of a genetic multicolor cell labeling approach for neural circuit analysis in Drosophila

The assembly of functional neural circuits during development is pivotal for the ability of the brain to generate complex behaviors. To facilitate the analysis of the underlying molecular mechanisms in Drosophila, we have developed a genetic multicolor cell labeling approach called Flybow (FB), which is based on the vertebrate Brainbow-2 system. FB relies on the stochastic expression of membrane tethered fluorescent proteins (FPs). FP encoding sequences were arranged in pairs within one or two cassettes each flanked by recombination sites. Recombination mediated by an inducible modified Flp/FRT system results in both excisions and inversions of the flanked cassettes providing temporal control of FP expression. Moreover, FB employs the GAL4/UAS system and hence can be used to investigate distinct cell populations in the tissue of interest. We have generated three FB variants. FB 1.0 consists of one cassette driving expression of either mCherry or V5-tagged Cerulean. FB 1.1 contains a second cassette with opposing enhanced green fluorescent protein (EGFP) and mCitrine cDNAs leading to stochastic expression of four FPs. Finally, FB2.0 contains an additional excisable cassette flanked by classical FRT sites to refine transgene expression in specific cell types, in which Gal4 and Flp activities overlap. The FB approach was validated by investigating neural circuit assembly and connectivity in the visual system. FB makes it possible to visualize dendritic and axonal arborizations of different neuron subtypes and the morphology of glial cells with single cell resolution in one sample. Using live and fixed embryonic tissue, we could show that FB is suitable for studies of this early developmental stage. Additionally, we demonstrated that the approach can be used in non-neural tissues. Finally, combining the mosaic analysis with a repressible cell marker (MARCM) and FB approaches, we demonstrate that our technique is compatible with available Drosophila tools for genetic dissection of neural circuit formation