Optimization-based interactive segmentation interface for multiregion problems.

Interactive segmentation is becoming of increasing interest to the medical imaging community in that it combines the positive aspects of both manual and automated segmentation. However, general-purpose tools have been lacking in terms of segmenting multiple regions simultaneously with a high degree of coupling between groups of labels. Hierarchical max-flow segmentation has taken advantage of this coupling for individual applications, but until recently, these algorithms were constrained to a particular hierarchy and could not be considered general-purpose. In a generalized form, the hierarchy for any given segmentation problem is specified in run-time, allowing different hierarchies to be quickly explored. We present an interactive segmentation interface, which uses generalized hierarchical max-flow for optimization-based multiregion segmentation guided by user-defined seeds. Applications in cardiac and neonatal brain segmentation are given as example applications of its generality

Serial optical coherence microscopy for label-free volumetric histopathology

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents

Nerve-targeted probes for fluorescence-guided intraoperative imaging.

A fundamental goal of many surgeries is nerve preservation, as inadvertent injury can lead to patient morbidity including numbness, pain, localized paralysis and incontinence. Nerve identification during surgery relies on multiple parameters including anatomy, texture, color and relationship to surrounding structures using white light illumination. We propose that fluorescent labeling of nerves can enhance the contrast between nerves and adjacent tissue during surgery which may lead to improved outcomes. Methods: Nerve binding peptide sequences including HNP401 were identified by phage display using selective binding to dissected nerve tissue. Peptide dye conjugates including FAM-HNP401 and structural variants were synthesized and screened for nerve binding after topical application on fresh rodent and human tissue and in-vivo after systemic IV administration into both mice and rats. Nerve to muscle contrast was quantified by measuring fluorescent intensity after topical or systemic administration of peptide dye conjugate. Results: Peptide dye conjugate FAM-HNP401 showed selective binding to human sural nerve with 10.9x fluorescence signal intensity (1374.44 ± 425.96) compared to a previously identified peptide FAM-NP41 (126.17 ± 61.03). FAM-HNP401 showed nerve-to-muscle contrast of 3.03 ± 0.57. FAM-HNP401 binds and highlight multiple human peripheral nerves including lower leg sural, upper arm medial antebrachial as well as autonomic nerves isolated from human prostate. Conclusion: Phage display has identified a novel peptide that selectively binds to ex-vivo human nerves and in-vivo using rodent models. FAM-HNP401 or an optimized variant could be translated for use in a clinical setting for intraoperative identification of human nerves to improve visualization and potentially decrease the incidence of intra-surgical nerve injury

Labeling of immune cells for in vivo monitoring of cell migration using magnetic resonance imaging and near-infrared imaging

In addition to macrophage localization, the opening of the blood brain barrier (BBB) as well as demyelination processes have been measured by MRI mapping gadolinium (Gd) related signal enhancement and magnetic transfer (MT), respectively. By successively applying this protocol at different time points during the EAE model progression, we were able to analyse the interdependence of immuno-cellular processes leading to axonal damage as well as the longitudinal evolution of pathological hallmarks of EAE. Furthermore, these techniques have been used to validate and quantify the anti-inflammatory effect of EDG-1 inhibitor FTY720 on EAE symptoms. Repeated USPIO administrations and MRI measurements combining the analysis of MT ratios and Gd-enhancement have been performed on vehicle and FTY720 treated animals. This study demonstrates that FTY720 can prevent inflammatory events in EAE rats by sequestrating immune cells in lymphoid organs during acute inflammation episodes. The third goal, was to translate the iron-labeling protocol from macrophages to T lymphocytes. As T-cells are initiators of the immune cascade leading to the occurence of symptoms in the EAE model, it would be highly relevant to visualize T lymphocytes prior to the onset of symptoms. Yet, as lymphocytes have no natural phagocytotic activity, in vivo tagging with CA was not feasible. We decided to label them in vitro with ferumoxides (FeO) and then, transfer iron-presenting cells adoptively to EAE animals intravenously. Different techniques have been used to evaluate the efficiency of lymphocytes labeling combining iron oxide particles with commonly available transfection agents (TAs) and the feasibility of labeling T lymphocytes in vitro has been demonstrated. However, the adoptive transfer of iron-tagged T-cells to EAE rats did not lead to the detection of these cells by MRI. As MR detection of iron-tagged cell in vivo was unsuccessful probably due to the inherent lack of sensitivity of the MRI technique for molecular changes and the dilution of labeled cells in the blood, we decided to switch to a more sensitive technique. Thus, the goal of the last part of the thesis was to label primary cultured T lymphocytes with a fluorescent dye: cyanine 5.5 (Cy5.5). The Tat peptide from the HIV virus chemically has been bound to the Cy5.5 to cargo the dye across T-cells membrane. The ability of this probe to penetrate T-cells and its potential toxicity has been evaluated in vitro. Subsequently, Cy5.5-Tat labeled lymphocytes were transferred to EAE rats in order to monitor their bio-distribution during EAE. Prominent signals have been obtained from rat brain and histological experimentation using confocal microscopy analysis have been performed to confirm the localization of Cy5.5 within the brain parenchyma

Monitoring of Immune Cell Response to B Cell Depletion Therapy and Nerve Root Injury Using Spio Enhanced MRI

Magnetic resonance (MR) is a robust platform for non-invasive, high-resolution anatomical imaging. However, MR imaging lacks the requisite sensitivity and contrast for imaging at the cellular level. This represents a clinical impediment to greater diagnostic accuracy. Recent advances have allowed for the in vivo visualization of populations and even of individual cells using superparamagnetic iron oxide (SPIO) MR contrast agents. These nanoparticles, commonly manifested as a core of a single iron oxide crystal or cluster of crystals coated in a biocompatible shell, function to shorten proton relaxation times. In MR imaging these constructs locally dephase protons, resulting in a decrease in signal (hypointensity) localized to the region of accumulation of SPIO. In the context of immune cell imaging, SPIO can provide insight into the cellular migration patterns, trafficking, temporal dynamics and progression of diseases and their related pathological states. Furthermore, by visualizing the presence and activity of immune cells, SPIO-enabled cellular imaging can help evaluate the efficacy of therapy in immune disorders. This thesis examines the production, modification and application of SPIO in a range of in vitro and in vivo immune-response-relevant cellular systems. The role of different nanoparticle characteristics including diameter, surface charge and concentration are investigated in the labeling of T cells in culture. Following optimization of SPIO loading conditions for lymphocytes, the effect these particles have on the activation of primary B cells are elucidated. B cells are tracked using a variety of modalities, with and without the application of B cell depleting therapy. This is to evaluate the efficacy of SPIO as in vivo marker for B cell distribution. Unmodified SPIO were applied to monitor macrophage infiltration in a transient nerve root compression model, with implications for neck pain diagnosis and treatment. Nanoparticle accumulation and MR hypointensity was correlated to the presence of activated macrophage at the site of injury. Taken together, the application of SPIO to study nanoparticle uptake in vitro and visualization of immune cells in vivo provide a basis for advanced study and diagnosis of diverse pathologies

Iron Labeling and Pre-Clinical MRI Visualization of Therapeutic Human Neural Stem Cells in a Murine Glioma Model

Treatment strategies for the highly invasive brain tumor, glioblastoma multiforme, require that cells which have invaded into the surrounding brain be specifically targeted. The inherent tumor-tropism of neural stem cells (NSCs) to primary and invasive tumor foci can be exploited to deliver therapeutics to invasive brain tumor cells in humans. Use of the strategy of converting prodrug to drug via therapeutic transgenes delivered by immortalized therapeutic NSC lines have shown efficacy in animal models. Thus therapeutic NSCs are being proposed for use in human brain tumor clinical trials. In the context of NSC-based therapies, MRI can be used both to non-invasively follow dynamic spatio-temporal patterns of the NSC tumor targeting allowing for the optimization of treatment strategies and to assess efficacy of the therapy. Iron-labeling of cells allows their presence to be visualized and tracked by MRI. Thus we aimed to iron-label therapeutic NSCs without affecting their cellular physiology using a method likely to gain United States Federal Drug Administration (FDA) approval.For human use, the characteristics of therapeutic Neural Stem Cells must be clearly defined with any pertubation to the cell including iron labeling requiring reanalysis of cellular physiology. Here, we studied the effect of iron-loading of the therapeutic NSCs, with ferumoxide-protamine sulfate complex (FE-Pro) on viability, proliferation, migratory properties and transgene expression, when compared to non-labeled cells. FE-Pro labeled NSCs were imaged by MRI at tumor sites, after intracranial administration into the hemisphere contralateral to the tumor, in an orthotopic human glioma xenograft mouse model.FE-Pro labeled NSCs retain their proliferative status, tumor tropism, and maintain stem cell character, while allowing in vivo cellular MRI tracking at 7 Tesla, to monitor their real-time migration and distribution at brain tumor sites. Of significance, this work directly supports the use of FE-Pro-labeled NSCs for real-time tracking in the clinical trial under development: "A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically modified Neural Stem Cells Expressing Escherichia coli Cytosine Deaminase for Treatment of Recurrent High-Grade Gliomas"