Towards increased efficiency and automation in fluorescence micrograph analysis based on hand-labeled data

Held CH. Towards increased efficiency and automation in fluorescence micrograph analysis based on hand-labeled data. Bielefeld: Universität Bielefeld; 2013.In the past decade, automation in fluorescence microscopy has strongly increased, particularly in regards to image acquisition and sample preparation, which results in a huge volume of data. The amount of time required for manual assessment of an experiment is hence mainly determined by the amount of time required for data analysis. In addition, manual data analysis is often a task with poor reproducibility and lack of objectivity. Using automated image analysis software, the time required for data analysis can be reduced while quality and reproducibility of the evaluation are improved. Most image analysis approaches are based on a segmentation of the image. By arranging several image processing methods in a so-called segmentation pipeline, and by adjusting all parameters, a broad range of fluorescence image data can be segmented. The drawback of available software tools is the long time required to calibrate the segmentation pipeline for an experiment, particularly for researchers with little knowledge of image processing. As a result, many experiments that could benefit from automated image analysis are still evaluated manually. In order to reduce the amount of time users have to spend in adapting automated image analysis software to their data, research was carried out on a novel image analysis concept based on hand-labeled data. Using this concept, the user is required to provide hand-labeled cells, based on which an efficient combination of image processing methods and their parameterization is automatically calibrated, without further user input. The development of a segmentation pipeline that allows high-quality segmentation of a broad range of fluorescence micrographs in short time poses a challenge. In this work, a three-stage segmentation pipeline consisting of exchangeable preprocessing, figure-ground separation and cell-splitting methods was developed. These methods are mainly based on the state of the art, whereas some of them represent contributions to this status. Discretization of parameters must be performed carefully, as a broad range of fluorescence image data shall be supported. In order to allow calibration of the segmentation pipeline in a short time, discretization with equidistant as well as nonlinear step sizes was implemented. Apart from parameter discretization, quality of the calibration strongly depends on choice of the parameter optimization technique. In order to reduce calibration runtime, exploratory parameter space analysis was performed for different segmentation methods. This experiment showed that parameter spaces are mostly monotonous, but also show several local performance maxima. The comparison of different parameter optimization techniques indicated that the coordinate descent method results in a good parameterization of the segmentation pipeline in a small amount of time. In order to minimize the amount of time spent by the user in calibration of the system, correlation between the number of hand-labeled reference samples and the resulting segmentation performance was investigated. This experiment demonstrates that as few as ten reference samples often result in a good parameterization of the segmentation pipeline. Due to the low number of cells required for automatic calibration of the segmentation pipeline, as well as its short runtime, it can be concluded that the investigated method improves automation and efficiency in fluorescence micrograph analysis

A molecular atlas of cell types and zonation in the brain vasculature

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.Peer reviewe

Biological image analysis

In biological research images are extensively used to monitor growth, dynamics and changes in biological specimen, such as cells or plants. Many of these images are used solely for observation or are manually annotated by an expert. In this dissertation we discuss several methods to automate the annotating and analysis of bio-images. Two large clusters of methods have been investigated and developed. A first set of methods focuses on the automatic delineation of relevant objects in bio-images, such as individual cells in microscopic images. Since these methods should be useful for many different applications, e.g. to detect and delineate different objects (cells, plants, leafs, ...) in different types of images (different types of microscopes, regular colour photographs, ...), the methods should be easy to adjust. Therefore we developed a methodology relying on probability theory, where all required parameters can easily be estimated by a biologist, without requiring any knowledge on the techniques used in the actual software. A second cluster of investigated techniques focuses on the analysis of shapes. By defining new features that describe shapes, we are able to automatically classify shapes, retrieve similar shapes from a database and even analyse how an object deforms through time

Examining Uptake of Nanomaterials by Eukaryotic Cells with Digital Image Cytometry

Due to their small size and related interesting properties, artificial nanoma-terials are utilized for a great number of biological and medical applications. Cell entry routes, intracellular trafficking and processing of nanoparticles, which determine their fate, efficiency, and toxicity, are depending on various parameters of the specific nanomaterial, such as size, surface charge, surface chemistry and elasticity. Nanoparticle-cell interactions are typically elucidated by means of fluorescence microscopy. Cell functions can be observed by a multiplicity of commercially available probes. For the quantification of cell features from images (image cytometry), computer-based algorithms are favoured to avoid bias introduced by the subjective perception of the observer. By applying high throughput microscopy in combination with digital image cytometry the screening of high numbers of cells is made possible. With the large quantity of obtained data, cell populations can be identified and, in general, results that are statistically meaningful are obtained. In the first part of this work this method is applied in order to examine the cellular responses upon exposure to plasmonic poly(methacrylic acid)-coated gold nanoparticles (Au NPs) with respect to morphology and viability of human endothelial and epithelial cells (HUVECs and HeLa cells). Au NPs of 4-5 nm size were chosen which had been thoroughly characterized in terms of their physico-chemical parameters. These particles bear interesting properties for biomedical applications and, for several years, have been in the focus of research. In this work significant impacts on mitochondrial and lysosomal morphology upon exposure to the Au NPs are reported. The alteration of the structure of the cytoskeleton and a dramatically reduced proliferation are described. Interestingly, the smallest dose inducing the described cellular responses was of one or two magnitudes lower than those, where acute cytotoxicity and an increase in the production of reactive oxygen species (ROS) were observed. In the second part the process of endocytosis of polymer capsules is examined. These systems are seen as a promising tool for intracellular cargo delivery and release. After lipid raft-mediated phagocytosis, the capsules are transferred from the neutral extracellular medium to increasingly acidic intracellular vesicles. By embedding a pH-sensitive fluorescent dye into the cavity of the capsule the uptake process and the associated acidification can be monitored time-dependently. It is demonstrated that the kinetic of the acidification process strongly depends on the stiffness of the capsules. Soft particles with minor stiffness are transported faster into lysosomal structures than stiffer ones. Additionally, these sensor particles are used to confirm the importance of the V1G1-subunit of the vacuolar ATPase being responsible for vesicle acidification

Pharmacological Behavior of Systemically Administered Nanoparticles of Defined Properties: Mechanistic Investigations at the Organ, Tissue, and Cellular Levels

The objective of this thesis is to establish design rules for nanoparticle properties that enable their in vivo transport to target destinations. Gold nanoparticles containing surface-engrafted polyethylene glycol (PEG) chains are prepared with controlled physicochemical properties (hydrodynamic size, surface charge, targeting ligand density). Upon systemic injection into mice, the transport of nanoparticles is monitored by blood pharmacokinetics as well as distribution at the organ, tissue, and subcellular levels from the same injection in an individual animal. At a constant, slightly negative surface charge (ca. -10 mV), most PEGylated gold nanoparticles (PEG-AuNPs) deposit in the liver, spleen, and kidney of normal mice 24 hours after injection. Increasing retention in the liver (Kupffer cells) and spleen correlate positively with increasing nanoparticle diameter over the range of 25-165 nm, largely due to phagocytic uptake. Accumulation in the kidney is size-dependent, but shows a maximum uptake at ca. 75 ± 25 nm that also gives the highest deposition in the mesangium (uptake by mesangial cells). Tumor-bearing mice received injections of PEG-AuNPs of near-constant size (ca. 75 nm) and surface charge (ca. -10 mV) but with variable amounts of ligands that target cancer cells (0-144 ligands per nanoparticle). Independent of ligand content, nanoparticles accumulate in the tumor by the enhanced permeation and retention effect to the same magnitude, and adjacent to leukocytes. Nanoparticles only enter cancer cells in significant amounts when they contain targeting ligands above a threshold amount (between 18 and 144 ligands per nanoparticle). Mechanistic studies from model nanoparticles provide insights for the delivery of therapeutic nanoparticles. Systemic administrations of targeted, cyclodextrin-based, siRNA-containing nanoparticles are investigated in animals and humans (Phase I clinical trial). A fluorescent chemical stain with exposed adamantane molecules for binding into the cyclodextrin cups of the targeted nanoparticles is created, allowing for the examination of tumor tissue sections from animals and patient biopsies. Results from both animal and human tissues reveal intracellular, dose-dependent accumulation of targeted nanoparticles in cancer cells of the tumor.</p

Nanotechnology in Cancer Treatment

The collection is diverse in the topics covered as well as the contributors, who come from across the globe. The overall topic is inherently a complex one since it integrates the exceptional complexity of cancer biology and treatment with the large and rapidly growing number of potential nanostructures and modifications that could be applied to treatment. In both cancer and nanotechnology, a very small change of a relevant variable can generate exceptional variability in biological outcome, creating inherent challenges to clinical adoption. As with many high-potential fields in medicine, the recognition of exceptional potential for nanotechnology in cancer therapy, with associated expectations of rapid clinical impact, has been followed by disappointingly slow progress that highlights not failure but rather unrealistic expectations for rapid progress. We have passed that period of expected rapid clinical impact for nanoparticle treatment of cancer and are now well-entrenched in the more realistic phase of exploiting the easiest applications, in this case packaging of established chemotherapy drugs, while carefully studying the exceptional numbers of relevant variables that must be evaluated for efficacy and safety of innovative strategies. Although never as rapid as hoped, the progress is clear and clinical impact is growing

Exploration of carbon nanotube composites and piezoelectric materials for implantable devices

This thesis describes an exploration of carbon nanotube (CNT) nanocomposites for application in implantable medical devices. The focus here is on materials and structures of interest as components of devices incorporating electrodes. Electrodes for implantable devices are commonly required to interface between an electrical system, where the charge carriers are electrons presented through a metal, and human tissue, where the charge carriers are ions as well as electrons not in a metal. These interfaces are found to be prone to issues such as fibrosis and rejection. The properties of carbon nanomaterials, piezoelectric peptides/polymers and their composites suggest them as promising candidate materials that could resolve these issues. The superior conductivity, mechanical properties and chemical stability of carbon nanotubes have been explored in recent years for potential application in biomedical sensors and devices. This work has explored piezoelectric materials, carbon nanotubes, polymers and nanocomposites of these as potential components of implantable devices. Diphenylalanine is a chiral, amphiphilic dipeptide molecule which has the ability to self-assemble into piezoelectric microtubules. The self-assembly process of diphenylalanine microtubules has been explored and its properties have been compared to the properties of poly[vinylidenefluoride-co-trifluoroethylene] (P[VDF-TrFE]) electrospun nanofibres. Later parts of this work considered the deposition of electrodes by printing. The development of CNT-polymer nanocomposites as printable inks for the fabrication of electrodes was explored. The structure and properties of the piezoelectric nano/ micro-materials, CNT-peptide complex and conductive CNT-polymer printable inks were characterised by a range of microscopic and spectroscopic techniques. The viability of neural cells on the developed functional materials and electrodes were tested by metabolic activity measurements and immunochemical staining microscopy. A CNT-polymer ink demonstrated good conductivity and dimensional stability when printed by 3D printer. Good biocompatibility of all the functional materials developed have been demonstrated in vitro, showing promise for further development of soft electrodes and applications in nanostructure piezoelectric sensors and implantable devices

Polymer-Coated Inorganic Nanoparticles: Nanotools for Life Science Applications

This dissertaion focus on the synthesis, surface modification and characterization of inorganic nanoparticles(NPs), including magnetic, plasmonic and semiconductor NPs. With controlling the reaction conditions during the synthesis, different particle diameters in the range of 4 nm to 30 nm can be synthesized. Afterwards, polymer coating process was successfully applied to different materials by overcoating the NPs with an amphiphoilic polymer, which can make the particle water soluble. This work aimed to produce the polymer-­ coated nanoparticles,analyze and compare their physico-­‐chemical properties based on different materials,and further, to test their potential for different biological applications