Understanding and identifying critical protein-protein interactions is just one of the key outcomes in biological research. It can help to confirm key cellular interactions, which in some fields, such as cancer research, can result in a greater understanding of disease pathogenesis, elucidate mechanisms of therapeutic resistance and aid in the development of new specific targets, leading to new methods of prevention and treatment. Time-correlated single photon counting fluorescence lifetime imaging microscopy is just one of the tools used to carry out this line of research. Here we demonstrate a direct interaction between two proteins involved in gene regulation and expression; p21 and FMN2. Furthermore, we also show the capability of this system to measure chromatin compaction in three dimensions. However, fluorescence lifetime imaging has some drawbacks, acquisition times on such a system can range from the tens of seconds to minutes, which is often too long to comprehensively measure many biological events. But microscopy is always developing, aided by new techniques and, perhaps even more so, new technological developments. This thesis also demonstrates two new methods of light sheet microscopy, that use both new equipment made available because of technological developments to allow time resolved imaging and traditional microscopic aspects to form a light sheet system based on polarisation. It outlines the design and how to build these systems and presents their function to show their great promise. Both techniques presented in this thesis utilise aspects of light not conventionally used in light sheet microscopy. Further development of these systems and application of emerging technologies will yield a system capable of outperforming current light sheet fluorescence microscopy-based fluorescence lifetime imaging techniques. The implementation of polarisation control into such a system would enable three-dimensional anisotropy based SPIM-FLIM measurements, an indispensable tool in researching molecular orientation and mobility at a macroscopic level in developing organisms.

In many systems, in vitro or in vivo, it has become important to experimentally obtain dynamical information at many different positions simultaneously. This is a challenge as conventionally, dynamic information in biological systems is probed with a confocal microscope to perform either fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP), which can be damaging due to phototoxicity, and yields information at a single position. Advances in camera sensors have allowed their use in place of single point detectors and the implement of imaging FCS by way of single plane illumination microscopy (SPIM). In this modality, a light sheet with a thickness of only a few microns illuminates the sample and the fluorescence is projected orthogonally onto the camera chip. By imaging small regions of interest at a very high frame rate, we can determine dynamic parameters such as diffusion coefficients and local concentrations in a 2D array of pixels. In this thesis, I discuss the theoretical background, hardware setup, design and characterization of a SPIM which I have built in order to perform imaging FCS. / Thesis / Master of Science (MSc)

No description available.

Proper metazoan development requires that genes are expressed in a spatiotemporally controlled manner, with tightly regulated levels. Altering the expression of genes that govern development leads mostly to aberrations. However, alterations can also be beneficial, leading to the formation of new phenotypes, which contributes to the astounding diversity of animal forms. In the past the expression of developmental genes has been studied mostly in fixed tissues, which is unable to visualize these highly dynamic processes. We combine genomic fosmid transgenes, expressing genes of interest close to endogenous conditions, with Selective Plane Illumination Microscopy (SPIM) to image the expression of genes live with high temporal resolution and at single cell level in the entire embryo. In an effort to expand the toolkit for studying Drosophila development we have characterized the global expression patterns of various developmentally important genes in the whole embryo. To process the large datasets generated by SPIM, we have developed an automated workflow for processing on a High Performance Computing (HPC) cluster. In a parallel project, we wanted to understand how spatiotemporally regulated gene expression patterns and levels lead to different morphologies across Drosophila species. To this end we have compared by SPIM the expression of transcription factors (TFs) encoded by Drosophila melanogaster fosmids to their orthologous Drosophila pseudoobscura counterparts by expressing both fosmids in D. melanogaster. Here, we present an analysis of divergence of expression of orthologous genes compared A) directly by expressing the fosmids, tagged with different fluorophore, in the same D. melanogaster embryo or B) indirectly by expressing the fosmids, tagged with the same fluorophore, in separate D. melanogaster embryos. Our workflow provides powerful methodology for the study of gene expression patterns and levels during development, such knowledge is a basis for understanding both their evolutionary relevance and developmental function.

Proper metazoan development requires that genes are expressed in a spatiotemporally controlled manner, with tightly regulated levels. Altering the expression of genes that govern development leads mostly to aberrations. However, alterations can also be beneficial, leading to the formation of new phenotypes, which contributes to the astounding diversity of animal forms. In the past the expression of developmental genes has been studied mostly in fixed tissues, which is unable to visualize these highly dynamic processes. We combine genomic fosmid transgenes, expressing genes of interest close to endogenous conditions, with Selective Plane Illumination Microscopy (SPIM) to image the expression of genes live with high temporal resolution and at single cell level in the entire embryo. In an effort to expand the toolkit for studying Drosophila development we have characterized the global expression patterns of various developmentally important genes in the whole embryo. To process the large datasets generated by SPIM, we have developed an automated workflow for processing on a High Performance Computing (HPC) cluster. In a parallel project, we wanted to understand how spatiotemporally regulated gene expression patterns and levels lead to different morphologies across Drosophila species. To this end we have compared by SPIM the expression of transcription factors (TFs) encoded by Drosophila melanogaster fosmids to their orthologous Drosophila pseudoobscura counterparts by expressing both fosmids in D. melanogaster. Here, we present an analysis of divergence of expression of orthologous genes compared A) directly by expressing the fosmids, tagged with different fluorophore, in the same D. melanogaster embryo or B) indirectly by expressing the fosmids, tagged with the same fluorophore, in separate D. melanogaster embryos. Our workflow provides powerful methodology for the study of gene expression patterns and levels during development, such knowledge is a basis for understanding both their evolutionary relevance and developmental function.

A split-phase induction motor (SPIM) is obtained by splitting each of the three-phase stator windings of an induction motor into two equal halves. This results in two sets of three-phase windings with a spatial angle difference of 30◦ (electrical) between them. The two sets of windings are fed from two different voltage-source inverters for speed control of the split-phase motor drive. Low dc bus voltage requirement and improved torque profile are some of the advantages of the split-phase motor, compared to the normal three-phase induction motor. A pulse width modulation (PWM) technique is used to produce the gating signals for the power semiconductor devices in the two inverters. The PWM technique can either be a carrier comparison (CC) based method or a space-vector (SV) based scheme. The carrier based PWM methods employ six modulating waves, which are compared against a common triangular carrier to generate the gating pulses. In space-vector based PWM schemes, the voltage reference is specified in terms of a rotating reference vector. In each subcycle, a set of voltage vectors are applied for appropriate durations of time to produce an average vector equal to the reference vector. Unlike three-phase induction motor drives, where the voltage vectors are two dimensional, the voltage vectors in the case of SPIM drive are four dimensional. This thesis presents a detailed survey on carrier-comparison based and space-vector based PWM techniques for the SPIM drive. In this thesis, sine-triangle PWM (STPWM) is analyzed from a space-vector perspective. The set of voltage vectors applied and the sequence of application of the voltage vectors in each half-carrier cycle are studied. The analysis shows that the set of voltage vectors and the switching sequence employed by STPWM are different from those used by the well known SVPWM tech-niques. Two other CC based PWM techniques, based on common mode injection, are considered for the SPIM drive. In one method, the common-mode signal is derived from all the six modulating signals, and is the same for both the inverters. In the second method, the common-mode signal is different for the two inverters; each common-mode signal is derived from the three-phase sinusoidal signals of the respective inverter. The study shows that the latter method has the highest dc bus utilization and results in the lowest total harmonic distortion (THD) among the CC PWM techniques. An experimental comparison of the three carrier-comparison techniques with three well known space-vector PWM techniques is presented. Total harmonic distortion (THD) of the line current is measured at different modulation indices for all six techniques. The experimental results are obtained from a 6kW, 200V, 50Hz split-phase induction motor drive, with constant V /F ratio. The PWM techniques are implemented using an ALTERA cyclone II field programmable gate array (FPGA) digital controller. One of the SV techniques, termed here as 4-dimensional 24-sector (4D24SEC) PWM is found to be the best in terms of line current THD among all the CC and SV based PWM techniques considered. However, compared to any carrier-based technique, implementation of the 4D24SEC PWM based on the space vector approach is found to be resource intensive. Hence, an equivalent carrier-based implementation of 4D24SEC PWM is proposed in this thesis. The feasibility of the proposed approach is verified experimentally, and is found to be consuming much less logical resources than the space-vector implementation (i.e. 4102 logical elements for the CC approach as against 33,655 logical elements for the SV approach). A new space-vector PWM technique is also proposed in the thesis. This technique utilizes a new set of voltage vectors and a new switching sequence, which are motivated by the analyses of the carrier-based methods, presented earlier. The proposed technique is implemented, and is compared with other space-vector and carrier-based methods at different modulation indices and switching frequencies. The proposed PWM technique is found to have the same dc-bus utilization as the existing 4-dimensional SV based PWM techniques. The performance of the proposed method is found to be not better than existing 4-dimensional SV PWM methods. The possibilities for new switching sequence is being explored here.

Solid tumors are complex structures comprising besides the cancer cells vasculature, extracellular matrix (ECM), soluble molecules and a plethora of various other cell types. These components form a so-called tumour microenvironment. From the numerous cell types that are part of tumor microenvironment, tumor infiltrating lymphocytes (TILs) play a major role in patient prognosis. Their presence is also of major importance with regard to new biological therapies based on immune checkpoint inhibitors. Crucial role of TILs is also reflected by the new approaches in cancer diagnostics namely by Immunoscore method (currently used in clinical settings). Immunoscore is based on localization and quantification of CD3+ and CD8+ TILs in thin histological sections of tumor tissue. The question remains to which extent the information obtained from 2D slices reflects the situation in tumor microenvironment considering its spatial heterogeneity. The development of new methodological approaches allowing evaluation of histological information in 3D is the key to answer this question. The theoretical part of this work first describes the heterogeneity of the tumor microenvironment and the role of immune cells within it. Then, the role of spatial heterogeneity and its possible influence on the histopathological...

Multicellular life is formed by an orchestrated interplay of processes on different scales in space and time. Observing and quantitatively measuring these processes in an intact, living organism requires gentle and adaptive imaging. One example of such a process is the scaling of the mitotic spindle during early development. The spindle segregates the chromosomes during cell division and the spindle length determines the positioning of the chromosomes in the successive daughter cells. Thus, adaptation of spindle size to cell size is crucial for proper functioning. Early development is an excellent phase to study spindle scaling since cells rapidly divide in the absence of growth. In this phase, the spindle can be studied in cells of the same organism changing its volume orders of magnitude. During early zebrafish embryogenesis, the mitotic spindle only appears for three minutes out of the fifteen minutes cell cycle. Quantifying these short-lived events in a living embryo requires flexible and adaptive multi-resolution recordings, which are impossible with any state-of-the-art microscope. In this thesis, I present two new techniques to adaptively image biological samples based on light sheet fluorescence microscopy (LSFM). First, I present a remote, contact-free positioning technique based on magnetic forces to orient the sample in the microscope. When imaging biological samples, there is often only one sample orientation that offers the best view on the region of interest. This preferred orientation typically changes over time as the specimen grows and develops. The contact-free positioning technique allows to always image specimens from the optimal viewing angle. I demonstrate the functionality of this method by 3D orientation of zebrafish embryos and zebrafish larvae. Second, I present a new type of LSFM that autonomously adapts its detection scheme to the sample state. This microscope contains an adaptable magnification module to map the development of the millimeter-sized zebrafish embryo and measure single-molecule dynamics of individual spindles in a single experiment. To automatically adapt the detection scheme, I trained a Convolution Neural Network to detect the cell cycle state of individual cells from acquired fluorescence images. Using this new type of LSFM, I demonstrate autonomous measurements of the mitotic spindle scaling in freely developing zebrafish embryos. / Multizelluläres Leben wird durch ein orchestriertes Zusammenspiel von Prozessen auf verschiedenen Skalen in Raum und Zeit gebildet. Beobachtung und quantitative Messungen dieser Vorgänge in einem intakten, lebenden Organismus erfordern schonende und adaptive Bildgebung. Ein Beispiel für einen solchen Prozess ist die Größenanpassung der mitotischen Spindel während der frühen Entwicklung. Die Spindel trennt die Chromosomen während der Zellteilung und die Spindellänge bestimmt die Positionierung der Chromosomen in den Tochterzellen. Daher ist die Anpassung der Spindelgröße an die Zellgröße entscheidend für die ordnungsgemäße Funktion. Die Phase der frühen Entwicklung eignet sich hervorragend zur Untersuchung der Spindel-Skalierung, da die Zellen sich schnell teilen ohne zu wachsen. Während der frühen Zebrafischembryogenese erscheint die Spindel nur drei Minuten innerhalb des fünfzehnminütigen Zellzyklus. Die Quantifizierung dieser kurzlebigen Ereignisse in einem lebenden Embryo erfordert flexible und anpassungsfähige Aufnahmen mit variabler Auflösung, die mit keinem Mikroskop nach dem aktuellen Stand der Technik möglich sind. In dieser Arbeit präsentiere ich zwei neue Techniken zur adaptiven Abbildung biologischer Proben basierend auf der Lichtblatt-Fluoreszenzmikroskopie (LSFM). Zuerst stelle ich eine berührungslose Positionierungstechnik vor, die auf Magnetkräften basiert, um die Probe im Mikroskop zu orientieren. Bei der Abbildung biologischer Proben gibt es oft nur eine Probenorientierung, welche die beste Sicht auf die Region von Interesse bietet. Diese Vorzugsorientierung ändert sich typischerweise mit der Zeit, wenn die Probe wächst und sich entwickelt. Die Positionierungstechnik ermöglicht es, Proben immer aus dem optimalen Betrachtungswinkel abzubilden. Zweitens stelle ich einen neuen Typ von LSFM vor, der sein Detektionsschema autonom an den Probenzustand anpasst. Dieses Mikroskop enthält ein anpassbares Vergrößerungsmodul, um die Entwicklung des millimetergroßen Zebrafischembryos abzubilden und die Einzelmoleküldynamik einzelner Spindeln in einem einzigen Experiment zu messen. Um die Detektion automatisch anzupassen, trainierte ich ein Convolutional Neural Network, um den Zellzyklusstatus einzelner Zellen anhand der aufgenommenen Fluoreszenzbilder zu erkennen. Mit diesem neuen LSFM-Typ demonstriere ich autonome Messungen der Spindel-Skalierung in sich frei entwickelnden Zebrafischembryonen.

Η παρούσα διπλωματική εργασία πραγματεύεται τη μελέτη και κατασκευή ενός μετατροπέα για την οδήγηση ενός μονοφασικού επαγωγικού κινητήρα. Η εργασία αυτή εκπονήθηκε στο Εργαστήριο Ηλεκτρομηχανικής Μετατροπής Ενέργειας του Τμήματος Ηλεκτρολόγων Μηχανικών και Τεχνολογίας Υπολογιστών της Πολυτεχνικής Σχολής του Πανεπιστημίου Πατρών. Σκοπός είναι η μελέτη και κατασκευή ενός τριφασικού αντιστροφέα τάσης για τη λειτουργία και τον έλεγχο των στροφών ενός μονοφασικού επαγωγικού κινητήρα. Αρχικά μελετάται η βασική αρχή λειτουργίας του μονοφασικού επαγωγικού κινητήρα και αναλύονται οι τεχνικές εκκίνησης που χρησιμοποιούνται για σύνδεση του κινητήρα απευθείας στο δίκτυο. Ακόμα παρουσιάζονται τα ισοδύναμα κυκλώματα λειτουργίας του μονοφασικού επαγωγικού κινητήρα, η εξίσωση της ηλεκτρομαγνητικής ροπής του και προσομοιώνεται η λειτουργία του για τη μελέτη της στατικής συμπεριφοράς του. Στη συνέχεια γίνεται μια θεωρητική ανάλυση του κυκλώματος του τριφασικού αντιστροφέα τάσης που κατασκευάστηκε, καθώς και όλων των υπόλοιπων κυκλωμάτων που είναι αναγκαία για τη λειτουργία του. Επιπροσθέτως αναλύεται η μέθοδος παλμοδότησης των διακοπτικών στοιχείων του αντιστροφέα τάσης, που είναι η "Ημιτονοειδής Διαμόρφωση του Εύρους των Παλμών" (SPWM). Στο επόμενο βήμα αναλύονται τα τεχνικά χαρακτηριστικά όλων των κυκλωμάτων που κατασκευάστηκαν, και περιγράφεται ο κώδικας του προγράμματος του μικροελεγκτή, που χρησιμοποιήθηκε για την παραγωγή των παλμών. Τέλος, παραθέτουμε παλμογραφήματα και μετρήσεις που προέκυψαν από τα πειράματα που διενεργήθηκαν μετά την ολοκλήρωση της κατασκευής. / This diploma thesis discourse the analysis and construction of a converter topology for single phase induction motor drives. The project was based in the Laboratory of Electromechanical Energy Conversion of the department of Electrical and Computer Engineering of School Engineering of University of Patras. The objective of this project is the analysis and construction of a three phase voltage inverter to control the speed of a single phase induction motor. The first stage of this work is the study of the basic principle of operation of the single phase induction motor and the analysis of the starting techniques, used for the direct connection to the power grid. The equivalent circuits of the running single phase induction motor and the equation of the electromagnetic torque are also presented in this project. Following, there is a theoretical analysis of the three phase voltage inverter circuit, as well of all the remaining circuit, necessary for its function. Moreover the method of pulse generation for the switching elements of the voltage inverter is analyzed, which is the “Sinusoidal Pulse Width Modulation”. The next step is the analysis of the technical characteristics all of the circuits developed, as well the description of the program code for the microcontroller, used to produce the pulses. Finally oscillograph figures and measurements, occurred from the experiments transacted after the finalization of the construction, are adduced.