Towards a diffusion image processing validation and accuracy prediction framework

Validation is the main bottleneck preventing theadoption of many medical image processing algorithms inthe clinical practice. In the classical approach,a-posteriori analysis is performed based on someobjective metrics. In this work, a different approachbased on Petri Nets (PN) is proposed. The basic ideaconsists in predicting the accuracy that will result froma given processing based on the characterization of thesources of inaccuracy of the system. Here we propose aproof of concept in the scenario of a diffusion imaginganalysis pipeline. A PN is built after the detection ofthe possible sources of inaccuracy. By integrating thefirst qualitative insights based on the PN withquantitative measures, it is possible to optimize the PNitself, to predict the inaccuracy of the system in adifferent setting. Results show that the proposed modelprovides a good prediction performance and suggests theoptimal processing approach.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.

Forecasting the Earth"s radiation belts and modeling solar energetic particle events: Recent results from SPACECAST

High-energy charged particles in the van Allen radiation belts and in solar energetic particle events can damage satellites on orbit leading to malfunctions and loss of satellite service. Here we describe some recent results from the SPACECAST project on modelling and forecasting the radiation belts, and modelling solar energetic particle events. We describe the SPACECAST forecasting system that uses physical models that include wave-particle interactions to forecast the electron radiation belts up to 3 h ahead. We show that the forecasts were able to reproduce the >2 MeV electron flux at GOES 13 during the moderate storm of 7-8 October 2012, and the period following a fast solar wind stream on 25-26 October 2012 to within a factor of 5 or so. At lower energies of 10- a few 100 keV we show that the electron flux at geostationary orbit depends sensitively on the high-energy tail of the source distribution near 10 RE on the nightside of the Earth, and that the source is best represented by a kappa distribution. We present a new model of whistler mode chorus determined from multiple satellite measurements which shows that the effects of wave-particle interactions beyond geostationary orbit are likely to be very significant. We also present radial diffusion coefficients calculated from satellite data at geostationary orbit which vary with Kp by over four orders of magnitude. We describe a new automated method to determine the position at the shock that is magnetically connected to the Earth for modelling solar energetic particle events and which takes into account entropy, and predict the form of the mean free path in the foreshock, and particle injection efficiency at the shock from analytical theory which can be tested in simulations.

Diffusion av valsystem

Lectio praecursoria Åbo Akademi 1.2.2006

Shikonin-loaded antibody-armed nanoparticles for targeted therapy of ovarian cancer.

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.

Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.

In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼500nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB-SEM tomography, became available to image much larger volumes. During TEM tomography and FIB-SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples.

Forecasting the Earth"s radiation belts and modeling solar energetic particle events: Recent results from SPACECAST

High-energy charged particles in the van Allen radiation belts and in solar energetic particle events can damage satellites on orbit leading to malfunctions and loss of satellite service. Here we describe some recent results from the SPACECAST project on modelling and forecasting the radiation belts, and modelling solar energetic particle events. We describe the SPACECAST forecasting system that uses physical models that include wave-particle interactions to forecast the electron radiation belts up to 3 h ahead. We show that the forecasts were able to reproduce the >2 MeV electron flux at GOES 13 during the moderate storm of 7-8 October 2012, and the period following a fast solar wind stream on 25-26 October 2012 to within a factor of 5 or so. At lower energies of 10- a few 100 keV we show that the electron flux at geostationary orbit depends sensitively on the high-energy tail of the source distribution near 10 RE on the nightside of the Earth, and that the source is best represented by a kappa distribution. We present a new model of whistler mode chorus determined from multiple satellite measurements which shows that the effects of wave-particle interactions beyond geostationary orbit are likely to be very significant. We also present radial diffusion coefficients calculated from satellite data at geostationary orbit which vary with Kp by over four orders of magnitude. We describe a new automated method to determine the position at the shock that is magnetically connected to the Earth for modelling solar energetic particle events and which takes into account entropy, and predict the form of the mean free path in the foreshock, and particle injection efficiency at the shock from analytical theory which can be tested in simulations.

A connectome-based comparison of diffusion MRI schemes.

Diffusion MRI has evolved towards an important clinical diagnostic and research tool. Though clinical routine is using mainly diffusion weighted and tensor imaging approaches, Q-ball imaging and diffusion spectrum imaging techniques have become more widely available. They are frequently used in research-oriented investigations in particular those aiming at measuring brain network connectivity. In this work, we aim at assessing the dependency of connectivity measurements on various diffusion encoding schemes in combination with appropriate data modeling. We process and compare the structural connection matrices computed from several diffusion encoding schemes, including diffusion tensor imaging, q-ball imaging and high angular resolution schemes, such as diffusion spectrum imaging with a publically available processing pipeline for data reconstruction, tracking and visualization of diffusion MR imaging. The results indicate that the high angular resolution schemes maximize the number of obtained connections when applying identical processing strategies to the different diffusion schemes. Compared to the conventional diffusion tensor imaging, the added connectivity is mainly found for pathways in the 50-100mm range, corresponding to neighboring association fibers and long-range associative, striatal and commissural fiber pathways. The analysis of the major associative fiber tracts of the brain reveals striking differences between the applied diffusion schemes. More complex data modeling techniques (beyond tensor model) are recommended 1) if the tracts of interest run through large fiber crossings such as the centrum semi-ovale, or 2) if non-dominant fiber populations, e.g. the neighboring association fibers are the subject of investigation. An important finding of the study is that since the ground truth sensitivity and specificity is not known, the comparability between results arising from different strategies in data reconstruction and/or tracking becomes implausible to understand.

Long-term monitoring of post-stroke plasticity after transient cerebral ischemia in mice using in vivo and ex vivo diffusion tensor MRI.

WE USED A MURINE MODEL OF TRANSIENT FOCAL CEREBRAL ISCHEMIA TO STUDY: 1) in vivo DTI long-term temporal evolution of the apparent diffusion coefficient (ADC) and diffusion fractional anisotropy (FA) at days 4, 10, 15 and 21 after stroke 2) ex vivo distribution of a plasticity-related protein (GAP-43) and its relationship with the ex vivo DTI characteristics of the striato-thalamic pathway (21 days). All animals recovered motor function. In vivo ADC within the infarct was significantly increased after stroke. In the stroke group, GAP-43 expression and FA values were significantly higher in the ipsilateral (IL) striatum and contralateral (CL) hippocampus compared to the shams. DTI tractography showed fiber trajectories connecting the CL striatum to the stroke region, where increased GAP43 and FA were observed and fiber tracts from the CL striatum terminating in the IL hippocampus.Our data demonstrate that DTI changes parallel histological remodeling and recovery of function.

Monte Carlo study of a kinetic lattice model with random diffusion of disorder

We report Monte Carlo results for a nonequilibrium Ising-like model in two and three dimensions. Nearest-neighbor interactions J change sign randomly with time due to competing kinetics. There follows a fast and random, i.e., spin-configuration-independent diffusion of Js, of the kind that takes place in dilute metallic alloys when magnetic ions diffuse. The system exhibits steady states of the ferromagnetic (antiferromagnetic) type when the probability p that J>0 is large (small) enough. No counterpart to the freezing phenomena found in quenched spin glasses occurs. We compare our results with existing mean-field and exact ones, and obtain information about critical behavior.

Reaction-diffusion lattice gas: Theory and computer results

We report on the study of nonequilibrium ordering in the reaction-diffusion lattice gas. It is a kinetic model that relaxes towards steady states under the simultaneous competition of a thermally activated creation-annihilation $(reaction$) process at temperature T, and a diffusion process driven by a heat bath at temperature T?T. The phase diagram as one varies T and T, the system dimension d, the relative priori probabilities for the two processes, and their dynamical rates is investigated. We compare mean-field theory, new Monte Carlo data, and known exact results for some limiting cases. In particular, no evidence of Landau critical behavior is found numerically when d=2 for Metropolis rates but Onsager critical points and a variety of first-order phase transitions.

Mechanisms underlying functional recovery after in vivo focal cerebral ischemia : from preconditioning to diffusion MRI

Version abregée L'ischémie cérébrale est la troisième cause de mort dans les pays développés, et la maladie responsable des plus sérieux handicaps neurologiques. La compréhension des bases moléculaires et anatomiques de la récupération fonctionnelle après l'ischémie cérébrale est donc extrêmement importante et représente un domaine d'intérêt crucial pour la recherche fondamentale et clinique. Durant les deux dernières décennies, les chercheurs ont tenté de combattre les effets nocifs de l'ischémie cérébrale à l'aide de substances exogènes qui, bien que testées avec succès dans le domaine expérimental, ont montré un effet contradictoire dans l'application clinique. Une approche différente mais complémentaire est de stimuler des mécanismes intrinsèques de neuroprotection en utilisant le «modèle de préconditionnement» : une brève insulte protège contre des épisodes d'ischémie plus sévères à travers la stimulation de voies de signalisation endogènes qui augmentent la résistance à l'ischémie. Cette approche peut offrir des éléments importants pour clarifier les mécanismes endogènes de neuroprotection et fournir de nouvelles stratégies pour rendre les neurones et la glie plus résistants à l'attaque ischémique cérébrale. Dans un premier temps, nous avons donc étudié les mécanismes de neuroprotection intrinsèques stimulés par la thrombine, un neuroprotecteur «préconditionnant» dont on a montré, à l'aide de modèles expérimentaux in vitro et in vivo, qu'il réduit la mort neuronale. En appliquant une technique de microchirurgie pour induire une ischémie cérébrale transitoire chez la souris, nous avons montré que la thrombine peut stimuler les voies de signalisation intracellulaire médiées par MAPK et JNK par une approche moléculaire et l'analyse in vivo d'un inhibiteur spécifique de JNK (L JNK) .Nous avons également étudié l'impact de la thrombine sur la récupération fonctionnelle après une attaque et avons pu démontrer que ces mécanismes moléculaires peuvent améliorer la récupération motrice. La deuxième partie de cette étude des mécanismes de récupération après ischémie cérébrale est basée sur l'investigation des bases anatomiques de la plasticité des connections cérébrales, soit dans le modèle animal d'ischémie transitoire, soit chez l'homme. Selon des résultats précédemment publiés par divers groupes ,nous savons que des mécanismes de plasticité aboutissant à des degrés divers de récupération fonctionnelle sont mis enjeu après une lésion ischémique. Le résultat de cette réorganisation est une nouvelle architecture fonctionnelle et structurelle, qui varie individuellement selon l'anatomie de la lésion, l'âge du sujet et la chronicité de la lésion. Le succès de toute intervention thérapeutique dépendra donc de son interaction avec la nouvelle architecture anatomique. Pour cette raison, nous avons appliqué deux techniques de diffusion en résonance magnétique qui permettent de détecter les changements de microstructure cérébrale et de connexions anatomiques suite à une attaque : IRM par tenseur de diffusion (DT-IR1V) et IRM par spectre de diffusion (DSIRM). Grâce à la DT-IRM hautement sophistiquée, nous avons pu effectuer une étude de follow-up à long terme chez des souris ayant subi une ischémie cérébrale transitoire, qui a mis en évidence que les changements microstructurels dans l'infarctus ainsi que la modification des voies anatomiques sont corrélés à la récupération fonctionnelle. De plus, nous avons observé une réorganisation axonale dans des aires où l'on détecte une augmentation d'expression d'une protéine de plasticité exprimée dans le cône de croissance des axones (GAP-43). En appliquant la même technique, nous avons également effectué deux études, rétrospective et prospective, qui ont montré comment des paramètres obtenus avec DT-IRM peuvent monitorer la rapidité de récupération et mettre en évidence un changement structurel dans les voies impliquées dans les manifestations cliniques. Dans la dernière partie de ce travail, nous avons décrit la manière dont la DS-IRM peut être appliquée dans le domaine expérimental et clinique pour étudier la plasticité cérébrale après ischémie. Abstract Ischemic stroke is the third leading cause of death in developed countries and the disease responsible for the most serious long-term neurological disability. Understanding molecular and anatomical basis of stroke recovery is, therefore, extremely important and represents a major field of interest for basic and clinical research. Over the past 2 decades, much attention has focused on counteracting noxious effect of the ischemic insult with exogenous substances (oxygen radical scavengers, AMPA and NMDA receptor antagonists, MMP inhibitors etc) which were successfully tested in the experimental field -but which turned out to have controversial effects in clinical trials. A different but complementary approach to address ischemia pathophysiology and treatment options is to stimulate and investigate intrinsic mechanisms of neuroprotection using the "preconditioning effect": applying a brief insult protects against subsequent prolonged and detrimental ischemic episodes, by up-regulating powerful endogenous pathways that increase resistance to injury. We believe that this approach might offer an important insight into the molecular mechanisms responsible for endogenous neuroprotection. In addition, results from preconditioning model experiment may provide new strategies for making brain cells "naturally" more resistant to ischemic injury and accelerate their rate of functional recovery. In the first part of this work, we investigated down-stream mechanisms of neuroprotection induced by thrombin, a well known neuroprotectant which has been demonstrated to reduce stroke-induced cell death in vitro and in vivo experimental models. Using microsurgery to induce transient brain ischemia in mice, we showed that thrombin can stimulate both MAPK and JNK intracellular pathways through a molecular biology approach and an in vivo analysis of a specific kinase inhibitor (L JNK1). We also studied thrombin's impact on functional recovery demonstrating that these molecular mechanisms could enhance post-stroke motor outcome. The second part of this study is based on investigating the anatomical basis underlying connectivity remodeling, leading to functional improvement after stroke. To do this, we used both a mouse model of experimental ischemia and human subjects with stroke. It is known from previous data published in literature, that the brain adapts to damage in a way that attempts to preserve motor function. The result of this reorganization is a new functional and structural architecture, which will vary from patient to patient depending on the anatomy of the damage, the biological age of the patient and the chronicity of the lesion. The success of any given therapeutic intervention will depend on how well it interacts with this new architecture. For this reason, we applied diffusion magnetic resonance techniques able to detect micro-structural and connectivity changes following an ischemic lesion: diffusion tensor MRI (DT-MRI) and diffusion spectrum MRI (DS-MRI). Using DT-MRI, we performed along-term follow up study of stroke mice which showed how diffusion changes in the stroke region and fiber tract remodeling is correlating with stroke recovery. In addition, axonal reorganization is shown in areas of increased plasticity related protein expression (GAP 43, growth axonal cone related protein). Applying the same technique, we then performed a retrospective and a prospective study in humans demonstrating how specific DTI parameters could help to monitor the speed of recovery and show longitudinal changes in damaged tracts involved in clinical symptoms. Finally, in the last part of this study we showed how DS-MRI could be applied both to experimental and human stroke and which perspectives it can open to further investigate post stroke plasticity.

Effect of growth regulators on 'Brookfield' apple gas diffusion and metabolism under controlled atmosphere storage

The objective of this work was to evaluate the effect of growth regulators on gas diffusion and on metabolism of 'Brookfield' apple, and to determine their correlation with quality characteristics of fruit stored in controlled atmosphere. A completely randomized design was used with four replicates. After eight months of storage, the effects of water (control), aminoethoxyvinylglycine (AVG), AVG + ethephon, AVG + naphthaleneacetic acid (NAA), ethephon + NAA, sole NAA, 1-MCP, ethylene absorption by potassium permanganate (ABS), AVG + ABS, and of AVG + 1-MCP - applied at different rates and periods - were evaluated on: gas diffusion rate, ethylene production, respiratory rate, internal ethylene concentration, internal CO2 content, mealiness, and intercellular space. Fruit from the control and sole NAA treatments had the highest mealiness occurrence. Growth regulators significantly changed the gaseous diffusion through the pulp of 'Brookfield' apple, mainly in the treatment AVG + ABS, which kept the highest gas diffusion rate. NAA spraying in the field, with or without another growth regulator, increased ripening metabolism by rising ethylene production and respiration rate, and reduced gas diffusion during shelf life. AVG spraying cannot avoid the ethephon effect during the ripening process, and reduces both the internal space and mealiness incidence, but it is not able to induce ethylene production or to increase respiration rates.