Automated Vascular Smooth Muscle Segmentation, Reconstruction, Classification and Simulation on Whole-Slide Histology

Histology of the microvasculature depicts detailed characteristics relevant to tissue perfusion. One important histologic feature is the smooth muscle component of the microvessel wall, which is responsible for controlling vessel caliber. Abnormalities can cause disease and organ failure, as seen in hypertensive retinopathy, diabetic ischemia, Alzheimer’s disease and improper cardiovascular development. However, assessments of smooth muscle cell content are conventionally performed on selected fields of view on 2D sections, which may lead to measurement bias. We have developed a software platform for automated (1) 3D vascular reconstruction, (2) detection and segmentation of muscularized microvessels, (3) classification of vascular subtypes, and (4) simulation of function through blood flow modeling. Vessels were stained for α-actin using 3,3\u27-Diaminobenzidine, assessing both normal (n=9 mice) and regenerated vasculature (n=5 at day 14, n=4 at day 28). 2D locally adaptive segmentation involved vessel detection, skeletonization, and fragment connection. 3D reconstruction was performed using our novel nucleus landmark-based registration. Arterioles and venules were categorized using supervised machine learning based on texture and morphometry. Simulation of blood flow for the normal and regenerated vasculature was performed at baseline and during demand based on the structural measures obtained from the above tools. Vessel medial area and vessel wall thickness were found to be greater in the normal vasculature as compared to the regenerated vasculature (p\u3c0.001) and a higher density of arterioles was found in the regenerated tissue (p\u3c0.05). Validation showed: a Dice coefficient of 0.88 (compared to manual) for the segmentations, a 3D reconstruction target registration error of 4 μm, and area under the receiver operator curve of 0.89 for vessel classification. We found 89% and 67% decreases in the blood flow through the network for the regenerated vasculature during increased oxygen demand as compared to the normal vasculature, respectively for 14 and 28 days post-ischemia. We developed a software platform for automated vasculature histology analysis involving 3D reconstruction, segmentation, and arteriole vs. venule classification. This advanced the knowledge of conventional histology sampling compared to whole slide analysis, the morphological and density differences in the regenerated vasculature, and the effect of the differences on blood flow and function

Micro-computed tomographic analysis of the radial geometry of intrarenal artery-vein pairs in rats and rabbits: Comparison with light microscopy

We assessed the utility of synchrotron-radiation micro-computed tomography (micro-CT) for quantification of the radial geometry of the renal cortical vasculature. The kidneys of nine rats and six rabbits were perfusion fixed and the renal circulation filled with Microfil. In order to assess shrinkage of Microfil, rat kidneys were imaged at the Australian Synchrotron immediately upon tissue preparation and then post fixed in paraformaldehyde and reimaged 24 hours later. The Microfil shrank only 2-5% over the 24 hour period. All subsequent micro-CT imaging was completed within 24 hours of sample preparation. After micro-CT imaging, the kidneys were processed for histological analysis. In both rat and rabbit kidneys, vascular structures identified in histological sections could be identified in two-dimensional (2D) micro-CT images from the original kidney. Vascular morphology was similar in the two sets of images. Radial geometry quantified by manual analysis of 2D images from micro-CT was consistent with corresponding data generated by light microscopy. However, due to limited spatial resolution when imaging a whole organ using contrast-enhanced micro-CT, only arteries ≥100 and ≥60 μm in diameter, for the rat and rabbit respectively, could be assessed. We conclude that it is feasible and valid to use micro-CT to quantify vascular geometry of the renal cortical circulation in both the rat and rabbit. However, a combination of light microscopic and micro-CT approaches are required to evaluate the spatial relationships between intrarenal arteries and veins over an extensive range of vessel size

A Method for 3D Histopathology Reconstruction Supporting Mouse Microvasculature Analysis.

Structural abnormalities of the microvasculature can impair perfusion and function. Conventional histology provides good spatial resolution with which to evaluate the microvascular structure but affords no 3-dimensional information; this limitation could lead to misinterpretations of the complex microvessel network in health and disease. The objective of this study was to develop and evaluate an accurate, fully automated 3D histology reconstruction method to visualize the arterioles and venules within the mouse hind-limb. Sections of the tibialis anterior muscle from C57BL/J6 mice (both normal and subjected to femoral artery excision) were reconstructed using pairwise rigid and affine registrations of 5 µm-thick, paraffin-embedded serial sections digitized at 0.25 µm/pixel. Low-resolution intensity-based rigid registration was used to initialize the nucleus landmark-based registration, and conventional high-resolution intensity-based registration method. The affine nucleus landmark-based registration was developed in this work and was compared to the conventional affine high-resolution intensity-based registration method. Target registration errors were measured between adjacent tissue sections (pairwise error), as well as with respect to a 3D reference reconstruction (accumulated error, to capture propagation of error through the stack of sections). Accumulated error measures were lower (

Novel 3D Microscopic Analysis of Human Placental Villous Trees Reveals Unexpected Significance of Branching Angles

The villous trees of human placentas delineate the fetomaternal border and are complex three-dimensional (3D) structures. Thus far, they have primarily been analyzed as thin, two-dimensional (2D) histological sections. However, 2D sections cannot provide access to key aspects such as branching nodes and branch order. Using samples taken from 50 normal human placentas at birth, in the present study we show that analysis procedures for 3D reconstruction of neuronal dendritic trees can also be used for analyzing trees of human placentas. Nodes and their branches (e.g., branching hierarchy, branching angles, diameters, and lengths of branches) can be efficiently measured in whole-mount preparations of isolated villous trees using high-end light microscopy. Such data differ qualitatively from the data obtainable from histological sections and go substantially beyond the morphological horizon of such histological data. Unexpectedly, branching angles of terminal branches of villous trees varied inversely with the fetoplacental weight ratio, a widely used clinical parameter. Since branching angles have never before been determined in the human placenta, this result requires further detailed studies in order to fully understand its impact

Investigation into the 3D structure of the developing human fetal heart

Investigating the developmental processes of the human fetal heart is a challenging task. Few reports describe the morphological features during the first stage of heart maturation and consecutive developmental periods after cardiogenesis. Reasons for this include the difficulty of collecting suitable samples and the limitations of investigating modalities. This research was proposed to clarify the detailed morphological features of normal human fetal hearts in early-stage maturation, using post-mortem samples. These samples were analysed using high-resolution episcopic microscopy (HREM), and compared with the latest clinical imaging taken by 3D fetal echocardiography and compared with mouse samples. HREM, a newly-developed high-quality image modality, produces a computerbased 3D reconstruction which enables us to visualize detailed spatial structures of small specimens. HREM includes several procedural steps, which may affect the histological or morphological structures of samples, so I explored the potential effects. I found 12% shrinkage due to dehydration and polymerization. Therefore, while the general appearance of 3D reconstructed images looked identical to the pictures of the original heart samples, it is important to consider the effects of shrinkage when interpreting the morphological assessment by HREM. Normal human fetal hearts from the 9th to 11th weeks of postmenstrual gestation demonstrated unique morphological findings. Ventricular walls and trabeculations showed thick and random cellular structures. Atrioventricular and semilunar valves were also thick but histological maturation was observed within a few weeks after cardiogenesis. The great arterial walls were thick and comprised of dense cellular matrix. Morphologically, several characteristic findings, such as large atrial appendages, the developmental process of formation of the membranous ventricular septum and prominent coronary arteries, were recognised during this period. Heart size increased linearly with gestation. Normal human fetal hearts demonstrate geometrical development and histological and morphological maturation after the period of cardiogenesis. In comparison with human fetal hearts, mouse hearts demonstrate dramatic morphological alterations during a short maturation period. Fetal mouse hearts show some similar morphological findings to the human fetal heart, such as large atrial appendages, lack of formation of the membranous septum, and thickened great arterial walls. This suggests a shared mechanism of fetal heart maturation in mammals. Detailed clinical information regarding cardiac morphology is vital for accurate prenatal heart diagnosis in the first trimester. Fetal echocardiography in early gestation has become routine practice. However, the technical limitations of image acquisition and picture resolution make it difficult to visualize clear 3D images for fetal cardiac diagnosis. Current modalities for clinical investigation by 3D echocardiography do not have sufficient resolution to enable detailed morphological investigation of the human fetal heart between 10th to 12th weeks of postmenstrual gestation. Only the original data of the four-chamber view demonstrated no offsetting of the atrioventricular valves as seen on HREM. Further technical advances in 3D echocardiography will be required to enable precise cardiac diagnosis in the first trimester. This thesis describes morphological development in normal human fetal hearts for the first few weeks after cardiogenesis and contributes to a better understanding of the normal appearances in the first trimester which is vital for future investigation into the origin of congenital heart disease

Does 2D-Histologic identification of villous types of human placentas at birth enable sensitive and reliable interpretation of 3D structure?

INTRODUCTION: The villous tree of human placentas is a complex three-dimensional (3D) structure which enables fetomaternal exchange. Current concepts of microscopic analyses are based on the analysis of two-dimensional (2D) histologic sections. For this approach, the assessment of the stromal core of sectioned villi is of key importance. The classification of stromal properties of sectioned villi allows allocation of villous sections to villous types which are named by their expected position in villous trees (terminal, intermediate, and stem villi). METHOD: The present study takes these current concepts of placental histology as hypothesis and validates them against predetermined 3D positions of branches of villous trees. The 3D positions were determined prior to histologic sectioning using a recently introduced 3D-microscopic approach. Individual histologic sections of villi were classified by their stromal structures and inter rater variability of these histologic assessments were determined. RESULTS/DISSCUSSION: Inter rater variability was high and indicates substantial observer influence on the outcome of histologic assessments. Cross-match of villous types with the predetermined positions of villous branches of villous trees revealed substantial mismatch between the outcome of stromal classification and 3D-position of the sectioned villi in the placental villous trees.DFG, grant numbers INST 86/1495-1 FUGG, INST86/1452-1 LAGG.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.placenta.2015.10.00

MicroCT of Coronary Stents: Staining Techniques for 3-D Pathological Analysis

In the area of translational research, stent developers consult pathologists to obtain the best and most complete amount of data from implanted test devices in the most efficient manner. Through the use of micron-scale computed tomography along with post-fixation staining techniques in this study, full volumes of previously implanted stents have been analyzed in-situ in a non-destructive manner. The increased soft tissue contrast imparted by metal-containing stains allowed for a qualitative analysis of the vessel’s response to the implant with greater sensitivity and specificity while reducing beam-hardening artifact from stent struts. The developed staining techniques included iodine-potassium iodide, phosphomolybdic acid, and phosphotungstic acid, all of which bind to soft tissue and improve image quality through their ability to attenuate high energy X-rays. With these stains, the overall soft tissue contrast increased by up to 85 percent and contrast between medial and neointimal layers of the vessel increased by up to 22 percent. Beam hardening artifact was also reduced by up to 38 percent after staining. Acquiring data from the entirety of the stent and the surrounding tissue increased the quality of stent analysis in multiple ways. The three dimensional data enabled a comprehensive analysis of stent performance, lending information such as neointimal hyperplasia, percent stenosis, delineation of vessel wall layers, stent apposition, and stent fractures. By providing morphological data about stent deployment and host response, this method circumvents the need to make the more traditional histology slides for a morphometric analysis. These same data may also be applied to target regions of interest to ensure histology slides are cut from the optimal locations for a more in-depth analysis. The agents involved in such techniques are readily available in most pathology laboratories, are safe to work with, and allow for rapid processing of tissue. The ability to forego histology altogether or to highly focus what histology is performed on a vessel has the potential to hasten the development process of any coronary stent

Molecular imaging of drug transit through the blood-brain barrier with MALDI mass spectrometry imaging

Drug transit through the blood-brain barrier (BBB) is essential for therapeutic responses in malignant glioma. Conventional methods for assessment of BBB penetrance require synthesis of isotopically labeled drug derivatives. Here, we report a new methodology using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) to visualize drug penetration in brain tissue without molecular labeling. In studies summarized here, we first validate heme as a simple and robust MALDI MSI marker for the lumen of blood vessels in the brain. We go on to provide three examples of how MALDI MSI can provide chemical and biological insights into BBB penetrance and metabolism of small molecule signal transduction inhibitors in the brain – insights that would be difficult or impossible to extract by use of radiolabeled compounds

Probing the Unseen Depths of the Hepatic Microarchitecture via Multimodal Microscopy

Multimodal microscopy combines the advantages and strengths of different imaging modalities in order to holistically characterise the organisation of biological organisms and their comprising constituents under healthy and diseased conditions, down to the spatial resolution required to understand the morphology and function of such structures. Given the profound advantages conferred by such an approach, this work broadly aimed to develop and exploit various multimodal and multi-dimensional imaging modalities in a complimentary, combined and/or correlative manner – namely, three-dimensional scanning electron microscopy, transmission electron tomography, bright-field light microscopy, confocal laser scanning microscopy and X-ray micro-computed tomography – in order to characterise and collect new information on the normal and pathological microarchitecture of rodent and human liver tissue in 3-D under various experimental conditions. The data reported in this work includes a comparative analysis of a variety of sample preparation protocols applied to rat liver tissue to determine the suitability of such protocols for the application of serial block-face scanning electron microscopy (SBF-SEM). Next, 3-D modelling and morphometric analysis (utilising the premier SBF-SEM protocol) was performed in order to visualise and quantify key features of the hepatic microarchitecture. We further outline a large-volume correlative light and electron microscopy approach utilising selective molecular probes for confocal laser scanning microscopy (actin, lipids and nuclei), combined with the 3-D ultrastructure of the same structures of interest, as revealed by SBF-SEM (Chapter 2). Development of a straightforward combinatorial sample preparation approach, followed by a swift multimodal imaging approach – combining X-ray micro-computed tomography, bright-field light microscopy and serial section scanning electron microscopy – facilitated the cross correlation of structure-function information on the same sample across diverse length scales (Chapter 3). Next, we outline a novel “silver filler pre-embedding approach” in order to reduce artefactual charging, minimise dataset acquisition time and improve resolution and contrast in rat liver tissue prepared for SBF-SEM (Chapter 4). Next, we employ a complementary imaging approach involving serial section scanning electron microscopy and transmission electron tomography in order to comparatively analyse the structure and morphometric parameters of thousands of normal- and giant mitochondria in human patients diagnosed with non-alcoholic fatty liver disease. In so doing, we reveal functional alterations associated with mitochondrial gigantism and propose a mechanism for their formation (Chapter 5). Finally, the significance of the results obtained, and major scientific advances reported in this work are discussed in-depth against the relevant literature. This is proceeded by the future outlooks and research that remains to be done, followed by the main conclusions of this Ph.D thesis (Chapter 6). In summary, our findings firmly establish the immense importance and value of contemporary multimodal microscopy modalities in modern life science research, for holistically revealing cellular structures along the vast length scales amongst which they exist, under healthy and clinically relevant pathological conditions

Development of three-dimensional, ex vivo optical imaging

The ability to analyse tissue in 3-D at the mesoscopic scale (resolution: 2-50 µm) has proven essential in the study of whole specimens and individual organs. Techniques such as ex vivo magnetic resonance imaging (MRI) and X-ray computed tomography (CT) have been successful in a number of applications. Although MRI has been used to image embryo development and gene expression in 3-D, its resolution is not sufficient to discriminate between the small structures in embryos and individual organs. Furthermore, since neither MRI nor X-ray CT are optical imaging techniques, none of them is able to make use of common staining techniques. 3-D images can be generated with confocal microscopy by focusing a laser beam to a point within the sample and collecting the fluorescent light coming from that specific plane, eliminating therefore out-of-focus light. However, the main drawback of this microscopy technique is the limited depth penetration of light (~1 mm). Tomographic techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (also known as single plane illumination microscopy, SPIM) are novel methods that fulfil a requirement for imaging of specimens which are too large for confocal imaging and too small for conventional MRI. To allow sufficient depth penetration, these approaches require specimens to be rendered transparent via a process known as optical clearing, which can be achieved using a number of techniques. The aim of the work presented in this thesis was to develop methods for threedimensional, ex vivo optical imaging. This required, in first instance, sample preparation to clear (render transparent) biological tissue. In this project several optical clearing techniques have been tested in order to find the optimal method per each kind of tissue, focusing on tumour tissue. Indeed, depending on its structure and composition (e.g. amount of lipids or pigments within the tissue) every tissue clears at a different degree. Though there is currently no literature reporting quantification of the degree of optical clearing. Hence a novel, spectroscopic technique for measuring the light attenuation in optically cleared samples is described in this thesis and evaluated on mouse brain. 5 Optical clearing was applied to the study of cancer. The main cancer model investigated in this report is colorectal carcinoma. Fluorescently labelled proteins were used to analyse the vascular network of colorectal xenograft tumours and to prove the effect of vascular disrupting agents on the vascular tumour network. Furthermore, optical clearing and fluorescent compounds were used for ex vivo analysis of perfusion of a human colorectal liver metastasis model