Mesoscopic 3D Quantitative Imaging: Attenuation Correction with OPTiSPIM

This thesis covers a number of technical developments, to improve mesoscopic imaging and analysis, with a focus on quantitative imaging. The main topic is attenuation in light sheet fluorescence microscopies (LSFM). Attenuation can be a prominent part of signal deterioration, however none of the presented technical improvements in LSFM address this problem directly. We address the problem by fusing optical projection tomography (OPT) with selective plane illumination microscopy (SPIM) in a hybrid setup (OPTiSPIM). In contrast to SPIM, that relys on fluorescent contrast, OPT can reconstruct data from absorbing contrast. Apart from having registered multimodal datasets by fusing data from different contrast mechanisms, the reconstructed absorption contrast provides a 3D attenuation map to the sample. This map is not accessible with a single SPIM scan, but allows for correcting the deterioration of the fluorescent SPIM signal. The corrected signal provides a more accurate measure of fluorescence in the sample.Aquesta tesi cobreix una sèrie de desenvolupaments tècnics, per a millorar l’adquisició i l’anàlisi d’imatges mesoscòpiques, centrant-se en l’obtenció quantitativa d’imatges. El tema principal és l’atenuació en microscòpia de làmina de llum fluorescent (LSFM). L’atenuació pot ser una causa important en el deteriorament de la senyal, tot i que cap de les millores tècniques existents en LSFM anaven adreçades a aquest problema directament. Hem abordat aquest problema mitjançant la fusió de la tomografia de projecció òptica (OPT) amb la microscòpia de pla d’il.luminació selectiva (SPIM) en un únic aparell híbrid (OPTiSPIM). A diferència de l’SPIM, que es basa en contrast de fluorescència, l’OPT permet recontruir dades del contrast d’absorbància. A part d’haver registrat dades multimodals mitjançant la fusió de dades de diferents mecanismes de contrast, el contrast d’absorció reconstruït obté un mapa d’atenuació en 3D de la mostra que permet la correcció del deteriorament de la senyal fluorescent obtinguda amb l’SPIM. Aquesta senyal corregida permet una mesura més acurada de la fluorescència de la mostra

Mesoscopic 3D Quantitative Imaging: Attenuation Correction with OPTiSPIM

This thesis covers a number of technical developments, to improve mesoscopic imaging and analysis, with a focus on quantitative imaging. The main topic is attenuation in light sheet fluorescence microscopies (LSFM). Attenuation can be a prominent part of signal deterioration, however none of the presented technical improvements in LSFM address this problem directly. We address the problem by fusing optical projection tomography (OPT) with selective plane illumination microscopy (SPIM) in a hybrid setup (OPTiSPIM). In contrast to SPIM, that relys on fluorescent contrast, OPT can reconstruct data from absorbing contrast. Apart from having registered multimodal datasets by fusing data from different contrast mechanisms, the reconstructed absorption contrast provides a 3D attenuation map to the sample. This map is not accessible with a single SPIM scan, but allows for correcting the deterioration of the fluorescent SPIM signal. The corrected signal provides a more accurate measure of fluorescence in the sample.Aquesta tesi cobreix una sèrie de desenvolupaments tècnics, per a millorar l’adquisició i l’anàlisi d’imatges mesoscòpiques, centrant-se en l’obtenció quantitativa d’imatges. El tema principal és l’atenuació en microscòpia de làmina de llum fluorescent (LSFM). L’atenuació pot ser una causa important en el deteriorament de la senyal, tot i que cap de les millores tècniques existents en LSFM anaven adreçades a aquest problema directament. Hem abordat aquest problema mitjançant la fusió de la tomografia de projecció òptica (OPT) amb la microscòpia de pla d’il.luminació selectiva (SPIM) en un únic aparell híbrid (OPTiSPIM). A diferència de l’SPIM, que es basa en contrast de fluorescència, l’OPT permet recontruir dades del contrast d’absorbància. A part d’haver registrat dades multimodals mitjançant la fusió de dades de diferents mecanismes de contrast, el contrast d’absorció reconstruït obté un mapa d’atenuació en 3D de la mostra que permet la correcció del deteriorament de la senyal fluorescent obtinguda amb l’SPIM. Aquesta senyal corregida permet una mesura més acurada de la fluorescència de la mostra

Attenuation artifacts in light sheet fluorescence microscopy corrected by OPTiSPIM

Light sheet fluorescence microscopy (LSFM) is rapidly becoming an essential technology for mesoscopic imaging of samples such as embryos and adult mouse organs. However, LSFM can suffer from optical artifacts for which there is no intrinsic solution. The attenuation of light due to absorbing material causes "shadow" artifacts along both the illumination and detection paths. Several approaches have been introduced to reduce this problem, including scanning illumination and multi-view imaging. However, neither of these approaches completely eliminates the problem. If the distribution of the absorbing material is complex, shadows cannot be avoided. We introduce a new approach that relies on multi-modal integration of two very different mesoscopic techniques. Unlike LSFM, optical projection tomography (OPT) can operate in transmission mode to create a voxel map of the 3D distribution of the sample's optical attenuation. Here, we demonstrate a hybrid instrument (OPTiSPIM) that can quantify this attenuation and use the information to correct the shadow artifacts of LSFM.The research was funded in part by the European Union’s 7th Framework VIBRANT project (No. 228933 of the FP7-NMP) and the Sinergia project (CRII3_125477) of the Swiss National Science Foundation (SNSF). We acknowledge support from the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017” and from the CERCA Programme/Generalitat de Catalunya

Quantitative measurements in 3-dimensional datasets of mouse lymph nodes resolve organ-wide functional dependencies

Deep tissue imaging has become state of the art in biology, but now the problem is to quantify spatial information in a global, organ-wide context. Although access to the raw data is no longer a limitation, the computational tools to extract biologically useful information out of these large data sets is still catching up. In many cases, to understand the mechanism behind a biological process, where molecules or cells interact with each other, it is mandatory to know their mutual positions. We illustrate this principle here with the immune system. Although the general functions of lymph nodes as immune sentinels are well described, many cellular and molecular details governing the interactions of lymphocytes and dendritic cells remain unclear to date and prevent an in-depth mechanistic understanding of the immune system. We imaged ex vivo lymph nodes isolated from both wild-type and transgenic mice lacking key factors for dendritic cell positioning and used software written in MATLAB to determine the spatial distances between the dendritic cells and the internal high endothelial vascular network. This allowed us to quantify the spatial localization of the dendritic cells in the lymph node, which is a critical parameter determining the effectiveness of an adaptive immune response.The authors are grateful for funding from VIBRANT Grant CP-IP 228933-2 from the FP7-NMP and Sinergia Grant CRSII3 125447 from the Swiss National Science Foundation (SNSF

Topologically selective islet vulnerability and self-sustained downregulation of markers for β-cell maturity in streptozotocin-induced diabetes

Mouse models of Streptozotocin (STZ) induced diabetes represent the most widely used preclinical diabetes research systems. We applied state of the art optical imaging schemes, spanning from single islet resolution to the whole organ, providing a first longitudinal, 3D-spatial and quantitative account of β-cell mass (BCM) dynamics and islet longevity in STZ-treated mice. We demonstrate that STZ-induced β-cell destruction predominantly affects large islets in the pancreatic core. Further, we show that hyperglycemic STZ-treated mice still harbor a large pool of remaining β-cells but display pancreas-wide downregulation of glucose transporter type 2 (GLUT2). Islet gene expression studies confirmed this downregulation and revealed impaired β-cell maturity. Reversing hyperglycemia by islet transplantation partially restored the expression of markers for islet function, but not BCM. Jointly our results indicate that STZ-induced hyperglycemia results from β-cell dysfunction rather than β-cell ablation and that hyperglycemia in itself sustains a negative feedback loop restraining islet function recovery.The authors thank Lars Haag and Lisa Sjöwall at Karolinska Institutet’s electron microscopy shared facility for providing TEM images. Dr. S. Willekens is acknowledged for help with editing of the manuscript. This project was funded by the Swedish Research Council, the Kempe foundations, Umeå University, Lenanders stiftelse, The strategic Research program in Diabetes at Karolinska Institutet, the Novo Nordisk Foundation, the Swedish Diabetes Association, the Family Knut and Alice Wallenberg Foundation, Diabetes Research and Wellness Foundation, the Stichting af Jochnick Foundation, the Family Erling-Persson Foundation, Berth von Kantzow’s Foundation, the Skandia Insurance Company, Ltd., ERC-2013-AdG 338936-BetaImage, the European Union’s Seventh Framework Program under grant agreements nos. 289932 and 613879