Correct layering (lamination) of neurons in the central nervous system (CNS) is critical for the tissue functionality. Neuronal lamination is established during development, when the majority of neurons have to move from their birthplace to the appropriate layer, where they function. Therefore, to grasp the logic of CNS development, it is essential to understand the kinetics and modes of the variety of neuronal translocation events. Most of our knowledge about neuronal translocation has been gained using fixed tissue or ex vivo imaging, which is not ideal for such a dynamic process heavily dependent on the surrounding environment. To avoid these limitations, I combined translucent zebrafish embryos with light sheet fluorescence microscopy, which together enabled gentle in toto imaging of neuronal translocation. I studied the translocation of retinal ganglion cells (RGCs) across the developing zebrafish retina. RGCs are the first neurons that differentiate in the vertebrate retina and are born in a proliferative zone at the retinal apical side. From here, they move basally, spanning the complete apico-basal length of the tissue. They are destined to occupy the most basal layer, where their axons form the optic nerve. Although it was described that RGCs move their soma while being attached to both apical and basal sides of the retina, the kinetics and cell biological mechanisms of somal translocation remained unknown. Extracting single cell behavior of RGCs from high-resolution movies of their translocation allowed for quantitative analysis of RGC movement. I revealed that RGCs cross the retina in less than two hours in a directionally persistent manner. The movement of RGC soma is a cell autonomously generated process, which requires intact microtubules and actin-dependent basal attachment of cells for speed and efficiency. Unexpectedly, interference with somal translocation leads to a shift towards a multipolar migratory mode, previously not observed for RGCs, in which they temporarily lose both apical and basal attachment and apico-basal polarity. The multipolar mode is overall slower and less directionally persistent, but still allows RGCs to reach the basal retina. However, when RGC translocation is inhibited completely, they differentiate ectopically in the center of the retina, which in turn triggers the formation of ectopic layers of later born neurons. These results highlight the importance of establishing the basal layer of ganglion cells for ensuing retinal lamination. Overall, I generated important advances in the understanding of neuronal translocation and lamination, which might be relevant for other parts of the CNS.

Chronic kidney disease is a progressive kidney disease that affects approximately one tenth of the world population. In almost all cases of progressive, end-stage kidney disease, fibrosis is seen. Renal fibrosis is a condition of the kidneys in which collagens and other proteins accumulate in the extracellular matrix of the kidneys. The aim of the project was to explore the use of Fast Green to visualize collagen in renal tissue using light-sheet microscopy. A healthy sample and a diseased sample of renal tissue was imaged in aqueous and anhydrous conditions and a comparison of the difference in staining pattern between the conditions was performed. There was a clear difference in staining pattern between the two conditions, where the samples prepared in anhydrous conditions showed a higher staining specificity to collagen as described previously for other types of tissue. An evaluation of the differences in collagen expression between the healthy and the diseased sample was performed as well. There was a visible difference between them where a higher expression of collagen was observed in numerous tubuli, indicative of pathological scarring. / Kronisk njursjukdom är en progressiv njursjukdom som påverkar approximativt en tiondel av världens befolkning. I alla progressiva njursjukdomar ses så kallad njurfibros. Njurfibros är ett tillstånd då kollagen och andra proteiner ansamlas i njurarnas extracellulära matris. Målet med projektet var att utforska användningen av Fast Green för att visualisera kollagen i njurvävnad med ”Light sheet”-mikroskopi. Vävnadsproverfrån frisk vävnad och sjuk vävnad avbildades i vattenhaltiga och vattenfria förhållanden och en jämförelse av inmärkningen mellan förhållandena utfördes. Det var en tydlig skillnad i inmärkningen mellan de två förhållandena, där det i de vattenfria proven observerades att Fast Green märkte in kollagen med högre specificitet. En jämförelse av skillnaderna i kollagenuttryck mellan det friska och det sjuka provet utfördes. Det var tydliga skillnader mellan proven, där ett stort antalkollagen-påverkade tubuli kunde observeras i det sjuka provet.

The cardiac conduction system is vital for the initiation and maintenance of the heartbeat. Over the recent years, the zebrafish (Danio rerio) has emerged as a promising model organism to study this specialized system. The embryonic zebrafish heart’s unique accessibility for light microscopy has put it in the focus of many cardiac researchers. However, imaging cardiac conduction in vivo remained a challenge. Typically, hearts had to be removed from the animal to make them accessible for fluorescent dyes and electrophysiology. Furthermore, no technique provided enough spatial and temporal resolution to study the importance of individual cells in the myocardial network. With the advent of light sheet microscopy, better camera technology, new fluorescent reporters and advanced image analysis tools, all-optical in vivo mapping of cardiac conduction is now within reach. In the course of this thesis, I developed new methods to image and manipulate cardiac conduction in 4D with cellular resolution in the unperturbed zebrafish heart. Using my newly developed methods, I could detect the first calcium sparks and reveal the onset of cardiac automaticity in the early heart tube. Furthermore, I could visualize the 4D cardiac conduction pattern in the embryonic heart and use it to study component-specific calcium transients. In addition, I could test the robustness of embryonic cardiac conduction under aggravated conditions, and found new evidence for the presence of an early ventricular pacemaker system. My results lay the foundation for novel, non-invasive in vivo studies of cardiac function and performance.

The understanding of the biological processes at the cellular and subcellular level requires the ability to directly visualize them. Fluorescence microscopy played a key role in biomedical imaging because of its high sensitivity and specificity. However, traditional fluorescence microscopy has a limited resolution due to optical diffraction. In recent years, various approaches have been developed to overcome the diffraction limit. Among these techniques, nonlinear structured illumination microscopy (SIM) has been demonstrated a fast and full field superresolution imaging tool, such as Saturated-SIM and Photoswitching-SIM. In this dissertation, I report a new approach that applies nonlinear structured illumination by combining a uniform excitation field and a patterned stimulated emission depletion (STED) field. The nature of STED effect allows fast switching response, negligible stochastic noise during switching, low shot noise and theoretical unlimited resolution, which predicts STED-SIM to be a better nonlinear SIM. After the algorithm development and the feasibility study by simulation, the STED-SIM microscope was tested on fluorescent beads samples and achieved full field imaging over 1 x 10 micron square at the speed of 2s/frame with 4-fold improved resolution. Our STED-SIM technique has been applied on biological samples and superresolution images with tubulin of U2OS cells and granules of neuron cells have been obtained. In this dissertation, an effort to apply a field enhancement mechanism, surface plasmon resonance (SPR), to nonlinear STED-SIM has been made and around 8 time enhancement on STED quenching effect was achieved. Based on this enhancement on STED, 1D SPR enhanced STED-SIM was built and 50 nm resolution of fluorescence beads sample was achieved. Algorithm improvement is required to achieve full field superresolution imaging with SPR enhanced STED-SIM. The application of nonlinear structured illumination in two photon light-sheet microscopy is also studied in this dissertation. Fluorescent cellular imaging of deep internal organs is highly challenging because of the tissue scattering. By combining two photon Bessel beam light-sheet microscopy and nonlinear SIM, 3D live sample imaging at cellular resolution in depth beyond 200 microns has been achieved on live zebrafish. Two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity.

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Selective plane illumination microscopy (SPIM) is becoming the method of choice for long-term 3D fluorescence imaging thanks to its low photo-toxicity and high imaging speed. However, SPIM is very data intensive: A single SPIM experiment can easily generate terabytes of image data, which is often overwhelming for biologists to handle. Moreover, large SPIM datasets often require additional computational power for processing. There is a lack of optimized analysis software to visualize and quantify such large datasets. As a result, the data size burden is limiting the accessibility of this immensely powerful technology. In this thesis, I investigated the root of the data burden in SPIM. I found that although the raw data volume generated by SPIM is large, the data product after processing is often very small in comparison. As a result, there are two ways of alleviating the data burden: the raw data to data product conversion ratio can be improved and the process of converting raw data to data product can be streamlined. In my Ph.D. project, I demonstrated three different approaches to tackle the data burden in SPIM. Firstly, I tested different lossless data compression methods on standard SPIM datasets that I collected during my thesis work. I found that integer compression algorithms are ideal for SPIM images. The data size can be reduced by more than half when compressed losslessly on-the-fly, reducing storage stress. Secondly, if the image quality could be improved, raw images would contain a higher amount of useful information and it would be less wasteful to store the large dataset. To illustrate this concept, I created a custom on-the-fly image analysis software that automatically selects the optimal imaging view in a multi-view SPIM experiment. By applying the workflow to zebrafish embryo imaging, I showcase that each multi-view dataset contains more information than in the conventional case. Moreover, it became possible to reduce the number of imaging views without compromising data quality. Lastly, raw data can also be converted into data product on-the-fly. The need to store raw images is often a result of the disconnect between imaging and data analysis. If the raw data can be analyzed in memory as soon as they are captured by the microscope, there is no need to keep the raw image data. I have built a custom image analysis pipeline to quantify zebrafish Rohon-Beard cells’ axon branching patterns. The image analysis software semi-automatically performs sample surface extraction and image unwrapping. The resultant dataset is a flat lateral view of the embryo. The processed dataset is less than 10% the size of the original images. I also show that through directionality analysis, the processed data can be used to identify wild type embryos from drug-treated samples. I also showcase a couple of other custom SPIM imaging workflows that I helped create. I have imaged patient-derived cancer spheroids and Xenopus oocytes in collaboration with other researchers. Here, the smart microscopy concept helped facilitate many data processing challenges involved. Overall, my thesis showcased that the data burden in SPIM can be addressed effectively by integrating image processing closely into the image capture process. I call this overall concept 'smart microscopy' and I believe it is the future of fluorescence microscopy.

The cardiac conduction system is vital for the initiation and maintenance of the heartbeat. Over the recent years, the zebrafish (Danio rerio) has emerged as a promising model organism to study this specialized system. The embryonic zebrafish heart’s unique accessibility for light microscopy has put it in the focus of many cardiac researchers. However, imaging cardiac conduction in vivo remained a challenge. Typically, hearts had to be removed from the animal to make them accessible for fluorescent dyes and electrophysiology. Furthermore, no technique provided enough spatial and temporal resolution to study the importance of individual cells in the myocardial network. With the advent of light sheet microscopy, better camera technology, new fluorescent reporters and advanced image analysis tools, all-optical in vivo mapping of cardiac conduction is now within reach. In the course of this thesis, I developed new methods to image and manipulate cardiac conduction in 4D with cellular resolution in the unperturbed zebrafish heart. Using my newly developed methods, I could detect the first calcium sparks and reveal the onset of cardiac automaticity in the early heart tube. Furthermore, I could visualize the 4D cardiac conduction pattern in the embryonic heart and use it to study component-specific calcium transients. In addition, I could test the robustness of embryonic cardiac conduction under aggravated conditions, and found new evidence for the presence of an early ventricular pacemaker system. My results lay the foundation for novel, non-invasive in vivo studies of cardiac function and performance.

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.

In biology, it is common to study cultured cells (in vitro) with fluorescence time-lapse microscopy. The cells are recorded for longer period of time and can later be viewed at an accelerated speed. During the acquisition some live cells tend to migrate. This can be a problem if the cell’s migration speed is high enough to move outside the field of view (FOV) during the acquisition time. The cells that moves outside the FOV can no longer be recorded and the information about them will be lost. This thesis presents scripts that have been developed for ZEN (blue) to be able to track a specific migrating cell of interest in real-time with automated control of imaging parameters. The microscope stage position is modified on-the-fly to have the tracked cell in the center of the FOV for the whole experiment. Three different types of experiments to track migrating NK cells were performed with the scripts. The results show that the scripts were able to track one NK cell for more than 1 hour in both conventional wide-field and lattice light-sheet microscopy. The segmentation was inaccurate when one or more objects were in close proximity to the tracked cell. By applying a watershed algorithm the segmentation result can be improved in some cases. / Inom cellulär biologi är det vanligt att studera odlade celler (in vitro) med time- lapse-mikroskopi. Flertals bilder tas på cellerna under en längre tidsperiod och när experimentet är klart så kan man titta på bilderna som en video. Under förvärvet av bilderna så tenderar vissa levande celler att migrera. Ett problem som kan uppstå är om cellens migrationshastighet är tillräckligt hög för att röra sig utanför synfältet under anskaffningstiden. De celler som rör sig utanför synfältet kan inte längre avbildas och informationen om dem kommer att gå förlorad. I denna avhandling presenteras programmeringskoder som har utvecklats för ZEN (blue) som kan spåra en specifik migrerande cell i realtid med automatiserad kontroll av bildbehandlings parametrar. Mikroskopets scenposition modifieras under experimentets gång för att få den spårade cellen kontinuerligt i mitten av synfältet. Tre olika sorters experiment i kombination med programmeringskoderna utfördes för att spåra NK-celler. Resultaten visar att programmeringskoderna lyckades spåra en NK-cell i mer än 1 timme i både ett bredfältsfluorescensmikroskop och ett lattice light-sheet mikroskop. Segmenteringen var felaktig när ett eller flera objekt var i närheten av den spårade cellen. Genom att tillämpa en watershed algoritm kan segmenteringsresultatet förbättras i vissa fall.