Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains

Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains

Super-Resolution Imaging of Subcortical White Matter using Stochastic Optical Reconstruction Microscopy (STORM) and Super-Resolution Optical Fluctuation Imaging (SOFI).

AIMS: The spatial resolution of light microscopy is limited by the wavelength of visible light (the "diffraction limit", approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter using STORM and SOFI. METHODS: Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled using standard immunohistochemical markers (neurofilament-H, myelin associated glycoprotein, GFAP). Image sequences were processed for STORM (effective pixel size 8-32 nm) and for SOFI (effective pixel size 80 nm). RESULTS: In human, rat and mouse subcortical white matter high quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. CONCLUSIONS: Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. [232 words] This article is protected by copyright. All rights reserved

Lung Imaging and Function Assessment using Non-Contrast-Enhanced Magnetic Resonance Imaging

Measurement of pulmonary ventilation and perfusion has significant clinical value for the diagnosis and monitoring of prevalent lung diseases. To this end, non-contrast-enhanced MRI techniques have emerged as a promising alternative to scintigraphical measurements, computed tomography, and contrast-enhanced MRI. Although these techniques allow the acquisition of both structural and functional information in the same scan session, they are prone to robustness issues related to imaging artifacts and post-processing techniques, limiting their clinical utilization. In this work, new acquisition and post-processing techniques were introduced for improving the robustness of non-contrast-enhanced MRI based functional lung imaging. Furthermore, pulmonary functional maps were acquired in 2-year-old congenital diaphragmatic hernia (CDH) patients to demonstrate the feasibility of non-contrast-enhanced MRI methods for functional lung imaging. In the first study, a multi-acquisition framework was developed to improve robustness against field inhomogeneity artifacts. This method was evaluated at 1.5T and 3T field strengths via acquisitions obtained from healthy volunteers. The results demonstrate that the proposed acquisition framework significantly improved ventilation map homogeneity p<0.05. In the second study, a post-processing method based on dynamic mode decomposition (DMD) was developed to accurately identify dominant spatiotemporal patterns in the acquisitions. This method was demonstrated on digital lung phantoms and in vivo acquisitions. The findings indicate that the proposed method led to a significant reduction in dispersion of estimated ventilation and perfusion map amplitudes across different number of measurements when compared with competing methods p<0.05. In the third study, the free-breathing non-contrast-enhanced dynamic acquisitions were obtained from 2-year-old patients after CDH repair, and then processed using the DMD to obtain pulmonary functional maps. Afterwards, functional differences between ipsilateral and contralateral lungs were assessed and compared with results obtained using contrast-enhanced MRI measurements. The results demonstrate that pulmonary ventilation and perfusion maps can be generated from dynamic acquisitions successfully without the need for ionizing radiation or contrast agents. Furthermore, lung perfusion parameters obtained with DMD MRI correlate very strongly with parameters obtained using dynamic contrast-enhanced MRI. In conclusion, the presented work improves the robustness and accuracy of non-contrast-enhanced functional lung imaging using MRI. Overall, the methods introduced in this work may serve as a valuable tool in the clinical adaptation of non-contrast-enhanced imaging methods and may be used for longitudinal assessments of pulmonary functional changes

Preclinical MRI of the Kidney

This Open Access volume provides readers with an open access protocol collection and wide-ranging recommendations for preclinical renal MRI used in translational research. The chapters in this book are interdisciplinary in nature and bridge the gaps between physics, physiology, and medicine. They are designed to enhance training in renal MRI sciences and improve the reproducibility of renal imaging research. Chapters provide guidance for exploring, using and developing small animal renal MRI in your laboratory as a unique tool for advanced in vivo phenotyping, diagnostic imaging, and research into potential new therapies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Preclinical MRI of the Kidney: Methods and Protocols is a valuable resource and will be of importance to anyone interested in the preclinical aspect of renal and cardiorenal diseases in the fields of physiology, nephrology, radiology, and cardiology. This publication is based upon work from COST Action PARENCHIMA, supported by European Cooperation in Science and Technology (COST). COST (www.cost.eu) is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation. PARENCHIMA (renalmri.org) is a community-driven Action in the COST program of the European Union, which unites more than 200 experts in renal MRI from 30 countries with the aim to improve the reproducibility and standardization of renal MRI biomarkers

Torsional wave elastography to assess the mechanical properties of the cornea

Corneal mechanical changes are believed to occur before any visible structural alterations observed during routine clinical evaluation. This study proposed developing an elastography technique based on torsional waves (TWE) adapted to the specificities of the cornea. By measuring the displacements in the propagation plane perpendicular to the axis of the emitter, the effect of guided waves in platelike media was proven negligible. Ex vivo experiments were carried out on porcine corneal samples considering a group of control and one group of alkali burn treatment ( NH 4OH) that modified the mechanical properties. Phase speed was recovered as a function of intraocular pressure (IOP), and a Kelvin-Voigt rheological model was fitted to the dispersion curves to estimate viscoelastic parameters. A comparison with uniaxial tensile testing with thin-walled assumptions was also performed. Both shear elasticity and viscosity correlated positively with IOP, being the elasticity lower and the viscosity higher for the treated group. The viscoelastic parameters ranged from 21.33 to 63.17 kPa, and from 2.82 to 5.30 Pa s, for shear elasticity and viscosity, respectively. As far as the authors know, no other investigations have studied this mechanical plane under low strain ratios, typical of dynamic elastography in corneal tissue. TWE reflected mechanical properties changes after treatment, showing a high potential for clinical diagnosis due to its rapid performance time and paving the way for future in vivo studies.Ministerio de Educacion, Cultura y Deporte Grant DPI2017-83859-R DPI2014-51870-R UNGR15-CE-3664 EQC2018-004508-PSpanish Government DTS15/00093 PI16/00339Instituto de Salud Carlos III y Fondos FederJunta de Andalucia PI-0107-2017 PIN-0030-2017 IE2017-5537MCIN/AEI - European Social Fund "Investing in your future" PRE2018-086085Consejeria de economia, conocimiento, empresas y universidad SOMM17/6109/UGR B-TEP-026- IE2017-5537 P18-RT-1653European Commission SOMM17/6109/UGR B-TEP-026- IE2017-5537 P18-RT-165

Doctor of Philosophy

dissertationIn Chapter 1, an introduction to basic principles or MRI is given, including the physical principles, basic pulse sequences, and basic hardware. Following the introduction, five different published and yet unpublished papers for improving the utility of MRI are shown. Chapter 2 discusses a small rodent imaging system that was developed for a clinical 3 T MRI scanner. The system integrated specialized radiofrequency (RF) coils with an insertable gradient, enabling 100 'm isotropic resolution imaging of the guinea pig cochlea in vivo, doubling the body gradient strength, slew rate, and contrast-to-noise ratio, and resulting in twice the signal-to-noise (SNR) when compared to the smallest conforming birdcage. Chapter 3 discusses a system using BOLD MRI to measure T2* and invasive fiberoptic probes to measure renal oxygenation (pO2). The significance of this experiment is that it demonstrated previously unknown physiological effects on pO2, such as breath-holds that had an immediate (<1 sec) pO2 decrease (~6 mmHg), and bladder pressure that had pO2 increases (~6 mmHg). Chapter 4 determined the correlation between indicators of renal health and renal fat content. The R2 correlation between renal fat content and eGFR, serum cystatin C, urine protein, and BMI was less than 0.03, with a sample size of ~100 subjects, suggesting that renal fat content will not be a useful indicator of renal health. Chapter 5 is a hardware and pulse sequence technique for acquiring multinuclear 1H and 23Na data within the same pulse sequence. Our system demonstrated a very simple, inexpensive solution to SMI and acquired both nuclei on two 23Na channels using external modifications, and is the first demonstration of radially acquired SMI. Chapter 6 discusses a composite sodium and proton breast array that demonstrated a 2-5x improvement in sodium SNR and similar proton SNR when compared to a large coil with a linear sodium and linear proton channel. This coil is unique in that sodium receive loops are typically built with at least twice the diameter so that they do not have similar SNR increases. The final chapter summarizes the previous chapters

3D Extrusion Printing of Biphasic Anthropomorphic Brain Phantoms Mimicking MR Relaxation Times Based on Alginate-Agarose-Carrageenan Blends

The availability of adapted phantoms mimicking different body parts is fundamental to establishing the stability and reliability of magnetic resonance imaging (MRI) methods. The primary purpose of such phantoms is the mimicking of physiologically relevant, contrast-creating relaxation times T1 and T2. For the head, frequently examined by MRI, an anthropomorphic design of brain phantoms would imply the discrimination of gray matter and white matter (WM) within defined, spatially distributed compartments. Multichannel extrusion printing allows the layer-by layer fabrication of multiple pastelike materials in a spatially defined manner with a predefined shape. In this study, the advantages of this method are used to fabricate biphasic brain phantoms mimicking MR relaxation times and anthropomorphic geometry. The printable ink was based on purely naturally derived polymers: alginate as a calcium-cross-linkable gelling agent, agarose, iota- carrageenan, and GdCl3 in different concentrations (0-280 mu mol kg-1) as the paramagnetic component. The suggested inks (e.g., 3Alg-1Agar-6Car) fulfilled the requirements of viscoelastic behavior and printability of large constructs (&gt;150 mL). The microstructure and distribution of GdCl3 were assessed by scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX). In closely monitored steps of technological development and characterization, from monophasic and biphasic samples as printable inks and cross-linked gels, we describe the construction of large-scale phantom models whose relaxation times were characterized and checked for stability over time