Automated procedures for quantification of rhizosphere physical properties in micro CT images

The rhizosphere, i.e. the zone of soil immediately surrounding plant roots plays a prominent role in supplying plants with water and nutrients. However, surprisingly little is known about rhizosphere physical properties and how they affect root growth, water and nutrient uptake. The lack of non-invasive and non-destructive imaging techniques necessary to observe living roots growing in undisturbed soil have been a main reason for this shortcoming. Recent advances in synchrotron X-ray micro tomography (CMT) provide the potential to directly observe soil physical properties around living roots in-situ.In this work we develop procedures for assisting scientist to study the soil properties by visualizing and automatically processing micro CT images. Specifically image de-noising in the wavelet domain is performed for convenient profiling and segmentation is applied for automated calculation of soil properties. As new measures we proposed the normalized radial and circular aggregation and water transportability and also have shown ways of generalizing the studies for 3D

와우이식 수술기주위 청각기능 평가를 위한 와우갑각 전기자극 유발 청성뇌간반응

학위논문(박사) -- 서울대학교대학원 : 자연과학대학 협동과정 뇌과학전공, 2021.8. 오승하.Abstract Promontory Electrically Evoked Auditory Brainstem Response for Perioperative Evaluation of Auditory Function in Cochlear Implant Doo Hee Kim Interdisciplinary Program in Neuroscience The Graduate School Seoul National University 서론: 인공와우는 보청기를 이용하여도 소리를 들을 수 없는 중고도 감각신경성 난청인을 위한 청각재활장치이다. 이 장치는 난청인의 달팽이관에 전극을 이식되어 소리정보가 담긴 전기자극으로 청각세포를 직접 자극하여 청력을 회복시켜준다. 인공와우 수술 후 예후예측을 위하여, 수술하기 전에 난청인의 청각계의 상태를 확인하는 것은 매우 중요하다. 전기자극 유발반응 관찰은 기존연구들을 통하여 유용성이 알려졌지만, 신뢰성 부족, 자극위치 확인 어려움, 단채널 전극으로 인한 제한적인 자극범위 등으로 임상적으로 거의 쓰이지 않고 있다. 본 연구에서는 와우갑각의 자극 위치변경에 따른 청신경 자극 유발 전위의 특성에 변화가 있는지 관찰하여, 와우갑각의 다채널 자극을 통한 청각기능평가의 필요성을 확인하였다. 방법: 동물의 와우갑각에 배치, 고정하기에 적합한 형태의 표면형 다채널 전극을 제작하고, 전기자극이 출력되는 신경자극장치를 개발하였다. 정상 청력 4마리, 난청 동물 4마리에 대하여 달팽이관의 뼈 벽인 와우갑각에 다채널 전극을 설치하여 달팽이관의 apex에 가까운 곳에 Ch-A, base에 가까운 곳에 Ch-B가 위치되도록 한다. 정상청력개체 3마리, 난청개체 2마리에 대하여 전극위치 확인을 위해 microCT촬영을 하였다. 두 개 채널을 이용하여 청신경을 전기자극하여 유발되는 전위 electrically evoked auditory brainstem response(EABR)를 측정하고, wave V의 amplitude growth function(AGF), slope, latency 등을 분석하였다. 정상청력, 난청, Ch-A자극시, Ch-B자극시의 4개 조합에 대하여 통계분석을 시행하였다. 정상청력, 난청동물의 달팽이관의 조직병리를 통하여 전기자극되어 청각계 활성에 기여하는 spiral ganglion neuron의 밀도 또한 비교하였다. 결과: 전극설치 후 microCT 촬영결과, 정상청력군 2마리는 전극이 와우갑각에 정위치하였고, 1마리는 정위치 하지 못하였다. 난청군은 1마리는 정위치하였고, 1마리는 와우갑각 표면에서 이격되어 있었다. AGF는 전극위치가 확인된 개체에서 청력에 상관없이 Ch-A가 Ch-B에 비하여 amplitude가 더 크고 기울기가 급한 구간이 많았다. Amplitude는 Ch-B에서 NH군이 deaf군에 비해 유의미하게 컸다. 각 군의 모든 개체에 대한 slope는 청력에 관계없이 Ch-A에서 Ch-B에 비하여 컸다. 정상청력군의 Ch-B가 난청군의 Ch-A에 비해 유의미하게 컸다. EABR이 관찰된 stimulus intensity의 범위는 Ch-A보다 Ch-B 자극의 경우 더 넓었다. Latency는 청력에 관계없이 Ch-A자극한 경우가 Ch-B자극한 경우에 비하여 유의미하게 짧았다. Ch-B에 대하여는 정상청력군이 난청군에 비하여 짧았고, 정상청력군의 Ch-A자극한 경우 난청군 Ch-B자극한 경우에 비하여 유의미하게 짧았다. Threshold는 유의미한 차이는 없었고, 단위면적당 spiral ganglion neuron의 밀도는 정상청력군이 난청군보다 약 2.9배 높았다. 결론: 동물 와우갑각 전기자극에 적합한 신경자극장치와 표면형 다채널전극을 개발하고 와우갑각에 전극을 고정한 후 EABR을 성공적으로 측정하였다. 와우갑각의 서로 다른 위치를 자극하기 위하여 두 채널을 자극하여 획득한 EABR의 AGF, slope 등의 특성에 차이가 있음을 확인하였다. 전극의 물리적 위치에 따라 전류자극되는 범위, 자극되는 나선신경절 세포의 개수와 분포에 따른 특성이 반영되었기 때문일 것으로 여겨지며, 임상에서 청각기능 평가를 위하여 promontory stimulation을 할 경우에는 다채널 전극을 이용한 평가의 필요성이 요구된다고 사료된다. 그러나 전극이 의도되지 않은 위치에 고정된 경우가 있어 추후 연구를 위하여는 전극배치와 고정방법의 개선 및 전극설치 후 electrode-tissue interface 상태 모니터 기능이 필요하다. 또한 partial hearing loss model에 대하여 와우갑각의 다양한 위치를 자극한 청신경 반응 특성의 관찰이 필요하다. 이를 통하여 인공와우 대상자에 대하여 수술기주위에 청신경 상태를 객관적으로 진단하고, 청각재활결과의 예후 예측에 활용을 기대할 수 있을 것이다. 핵심단어: 인공와우, 청각기능평가, 와우갑각자극, 다채널 전극, 전기유발 청성뇌간반응, 수술기주위, 예후예측 학번 : 2014-30097Abstract Promontory Electrically Evoked Auditory Brainstem Response for Perioperative Evaluation of Auditory Function in Cochlear Implant Doo Hee Kim Interdisciplinary Program in Neuroscience The Graduate School Seoul National University Introduction: A cochlear implant is a hearing rehabilitation device for moderate to severe sensorineural hearing loss who cannot hear the sound even with a hearing aid. This device restores hearing by directly stimulating the auditory nerve with electrical stimulation containing sound information by implanting electrodes into the cochlea of the deaf person. To predict the clinical outcome after cochlear implant surgery, it is imperative to check the auditory function of the deaf person before surgery. Although the usefulness of observing evoked responses by promontory stimulation to evaluate the auditory function has been known through previous studies, it is rarely used clinically due to lack of reliability, difficulty in identifying stimulation locations, and limited stimulation range by single-channel electrodes. In this study, the necessity of auditory function evaluation through multichannel stimulation of the promontory was confirmed by observing whether there was a change in the characteristics of the evoked potential by stimulation of the auditory nerve according to the change in the stimulation position of the promontory. Methods: A surface-type multichannel electrode suitable for placement on the promontory of an animal was designed, and a neural stimulator that outputs electrical stimulation was developed. For four normal hearing (NH) and four deaf animals, install multichannel electrodes on the promontory so that channel A(Ch-A) is located near the apex of the cochlea and channel B(Ch-B) is located near the base. MicroCT imaging was performed to confirm the electrode positions. Electrically evoked auditory brainstem response (EABR) induced by promontory stimulation was measured using the two electrode channels. The amplitude growth function (AGF), slope, and latency of wave V of EABR were analyzed. The density of spiral ganglion neurons (SGNs) contributing to auditory system activity by electrical stimulation through histology of the cochlea with NH and deafness was also compared. Results: It was confirmed by microCT scans that the electrode is positioned in the correct position on the promontory in 2 out of 3 animals in the NH group and 1 out of 2 in the deaf group. In the case of AGF, in subjects whose electrode positions were confirmed, there were many sections with greater amplitude and steeper slope during Ch-A stimulation than during Ch-B stimulation in both groups. The amplitude was significantly greater in the NH group than in the deaf group in Ch-B. The slope showed steeper in Ch-A than Ch-B in both groups. The range of stimulus intensities that EABR was observed was more comprehensive with Ch-B than Ch-A. The latency was significantly shorter with Ch-A than with Ch-B, regardless of hearing. In Ch-B, the latency of the NH group was shorter than that of the deaf group. The Ch-A stimulation of the NH group was significantly shorter than the Ch-B stimulation of the deaf group. There was no significant difference in the threshold. The density of SGN was about 2.9 times higher in the NH group than in the deaf group. Conclusion: The neural stimulation device and surface-type multichannel electrode suitable for electrical stimulation of the promontory in animals were developed, and the properties of the EABR with two channels in the normal hearing and deaf subjects were compared. It was confirmed that there were differences in characteristics of AGF, slope, and latency of EABR obtained by stimulating two channels to stimulate different positions of the promontory. This may be due to the characteristics of current spread, and the number and distribution of stimulated SGNs are reflected according to the physical location of the electrode. So, using multichannel electrodes is necessary for promontory stimulation for auditory function evaluation in clinical practice. However, there are cases where the electrode was fixed in an unintended position, so it is needed to improve the electrode design and fixation method and monitor the condition of the electrode-tissue interface after installation. In addition, it is necessary to observe the characteristics of the auditory nerve response that stimulated various positions of the promontory in the partial hearing loss model with the various distribution of spiral ganglion neurons. Through this, the auditory function of a person with hearing loss can be objectively evaluated. It is expected to be used as a perioperative prognostic factor in a cochlear implant. Keyword : Cochlear implant, evaluation of auditory function, promontory stimulation, multichannel electrode, electrically evoked auditory brainstem response, perioperative, prognostic factor Student Number : 2014-30097Chapter 1. Introduction 1 1.1. Study Background 1 1.2. Purpose of Research 7 Chapter 2. Materials and Methods 9 2.1. Development of a Neural Stimulator and electrode 9 2.1.1. Neural Stimulator 9 2.1.2. Surface Type Multichannel Electrode 11 2.2. Experimental Setup for EABR Recording 13 2.2.1. Animal Preparation 13 2.2.2. Electrode Position on the Promontory 16 2.2.3. Setup for EABR Recording 17 2.3. Data Processing and Analyzed Parameters 18 2.4. Histology 22 2.5. Statistical Analysis 23 Chapter 3. Results 24 3.1. Neural Stimulator and Electrode 24 3.1.1. Neural Stimulator 24 3.1.2. Surface Type Multichannel Electrode 28 3.2. Electrode Position on Promontory 29 3.3. Properties of EABR of Between Channel A and Channel B Stimulation 33 3.3.1. Comparison of Channel A and B in Normal Hearing Group 33 3.3.2. Comparison of Channel A and B in Deaf Group 34 3.4. Properties of EABR of Between Normal Hearing and Deaf 37 3.4.1. Comparison of AGF, Slope, Latency, and Threshold of in Normal Hearing and Deaf Group 37 3.4.2. Statistical Analysis of EABR Parameters of All Subjects in Normal Hearing and Deaf Group 39 3.4.3. Density of Spiral Ganglion Neuron 43 Chapter 4. Discussions 48 4.1. Comparison of Properties of EABR Between Channel A and Channel B 48 4.2. Comparison of Properties of EABR of Between Normal Hearing and Deaf 49 4.3. Clinical Implications 51 4.4. Limitation of This Work and Future Direction 53 4.4.1. Design and type of electrode, Electrode Installation, and Fixation 53 4.4.2. Condition of Electrode-Tissue Interface 54 4.4.3. Animal Deaf Model 55 Chapter 5. Conclusion 56 5.1. Summary and Clinical Implications 56 5.2. Limitations of This Study and Further Study 57 Acknowledgment 58 Bibliography 59 Abstract in Korean 72박

Imaging fascicular organisation in mammalian vagus nerve for selective VNS

Nerves contain a large number of nerve fibres, or axons, organised into bundles known as fascicles. Despite the somatic nervous system being well understood, the organisation of the fascicles within the nerves of the autonomic nervous system remains almost completely unknown. The new field of bioelectronics medicine, Electroceuticals, involves the electrical stimulation of nerves to treat diseases instead of administering drugs or performing complex surgical procedures. Of particular interest is the vagus nerve, a prime target for intervention due to its afferent and efferent innervation to the heart, lungs and majority of the visceral organs. Vagus nerve stimulation (VNS) is a promising therapy for treatment of various conditions resistant to standard therapeutics. However, due to the unknown anatomy, the whole nerve is stimulated which leads to unwanted off-target effects. Electrical Impedance Tomography (EIT) is a non-invasive medical imaging technique in which the impedance of a part of the body is inferred from electrode measurements and used to form a tomographic image of that part. Micro-computed tomography (microCT) is an ex vivo method that has the potential to allow for imaging and tracing of fascicles within experimental models and facilitate the development of a fascicular map. Additionally, it could validate the in vivo technique of EIT. The aim of this thesis was to develop and optimise the microCT imaging method for imaging the fascicles within the nerve and to determine the fascicular organisation of the vagus nerve, ultimately allowing for selective VNS. Understanding and imaging the fascicular anatomy of nerves will not only allow for selective VNS and the improvement of its therapeutic efficacy but could also be integrated into the study on all peripheral nerves for peripheral nerve repair, microsurgery and improving the implementation of nerve guidance conduits. Chapter 1 provides an introduction to vagus nerve anatomy and the principles of microCT, neuronal tracing and EIT. Chapter 2 describes the optimisation of microCT for imaging the fascicular anatomy of peripheral nerves in the experimental rat sciatic and pig vagus nerve models, including the development of pre-processing methods and scanning parameters. Cross-validation of this optimised microCT method, neuronal tracing and EIT in the rat sciatic nerve was detailed in Chapter 3. Chapter 4 describes the study with microCT with tracing, EIT and selective stimulation in pigs, a model for human nerves. The microCT tracing approach was then extended into the subdiaphragmatic branches of the vagus nerves, detailed in Chapter 5. The ultimate goal of human vagus nerve tracing was preliminarily performed and described in Chapter 6. Chapter 7 concludes the work and describes future work. Lastly, Appendix 1 (Chapter 8) is a mini review on the application of selective vagus nerve stimulation to treat acute respiratory distress syndrome and Appendix 2 is morphological data corresponding to Chapter 4

X-ray computed tomography

X-ray computed tomography (CT) can reveal the internal details of objects in three dimensions non-destructively. In this Primer, we outline the basic principles of CT and describe the ways in which a CT scan can be acquired using X-ray tubes and synchrotron sources, including the different possible contrast modes that can be exploited. We explain the process of computationally reconstructing three-dimensional (3D) images from 2D radiographs and how to segment the 3D images for subsequent visualization and quantification. Whereas CT is widely used in medical and heavy industrial contexts at relatively low resolutions, here we focus on the application of higher resolution X-ray CT across science and engineering. We consider the application of X-ray CT to study subjects across the materials, metrology and manufacturing, engineering, food, biological, geological and palaeontological sciences. We examine how CT can be used to follow the structural evolution of materials in three dimensions in real time or in a time-lapse manner, for example to follow materials manufacturing or the in-service behaviour and degradation of manufactured components. Finally, we consider the potential for radiation damage and common sources of imaging artefacts, discuss reproducibility issues and consider future advances and opportunities

Experimental imaging of asthma progression and therapeutic response in mouse lung models

Asthma ist eine Erkrankung die das komplette Immunsystems involviert, ein System so komplex, dass es sich nur unzureichend in-vitro studieren lässt. Daher haben sich Mausmodelle als ein unverzichtbares Werkzeug in der präklinischen Asthmaforschung etabliert. Da es sich weiterhin bei Asthma um eine Erkrankung handelt, die durch eine schnelle Änderung der Symptome gekennzeichnet ist, wäre longitudinale vorzugsweise nicht-invasive Bildgebung, insbesondere bei der Entwicklung und Bewertung neuer Therapiekonzepte von großem Interesse. Nachteilig hingegen ist, dass die Darstellung der Mauslunge in der Praxis auf Grund der Größe des Organs und, im Falle einer in vivo Bildgebung, durch die Bewegung des Brustkorbes sich als äußerst schwierig herausstellt. Die Vielzahl der Luft-Gewebe-Grenzflächen erzeugt starke Streuung in der optischen Bildgebung, der große Hohlraum der Lunge verursacht Suszeptibilitätsartefakte bei der MRT und die Rippen erschweren eine Ultraschallbildgebung. Aus diesen Gründen besteht ein großer Bedarf an neuen Bildgebungsverfahren, um die durch Asthma verursachten anatomischen, funktionalen und molekularen Veränderungen darstellen zu können. Um die Schwierigkeiten in der Lungenbildgebung bei Mäusen zu umgehen, habe ich mich auf drei wesentliche Bildgebungsstrategien fokussiert: A) anatomische Bildgebung durch “inline free propagation phase contrast computed tomography”, B) direkte Messung der Lungenfunktion durch “low dose planar cinematic x-ray imaging” und C) funktionale Bildgebung mit Hilfe der „near infrared fluorescence imaging“ in Kombination mit Antikörpern, die mit einem Fluoreszenzfarbstoff markiert wurden, oder “smart probes”, die in Gegenwart von Entzündungen aktiviert werden. Durch die Anwendung von “phase contrast computed tomography” für die anatomische Bildgebung war ich in der Lage morphologische Veränderung des Lungengewebes zu quantifizieren, indem ich lokal das Verhältnis zwischen Weichgewebe und Luft, das Zusammenziehen der Luftwege sowie das Anschwellen der Bronchialwände im asthmatischen Lungengewebe ausgewertet habe. Diese Parameter erlaubten es zwischen Mäusen von Asthmamodellen unterschiedlicher Schweregrade, therapierten und gesunden Mäusen zu unterscheiden. Zusätzlich ermöglichte diese Technik die Darstellung intra-tracheal applizierter Bariumsulfat markierter Makrophagen im Lungengewebe. Dies stellt meines Wissens die erste Kombination einer funktionalisierten Kontrastierung und hochauflösender Lungenbildgebung mittels CT unter in vivo ähnlichen Bedingungen dar. Um diese Ergebnisse mit dem Grad der asthmabedingten Kurzatmigkeit zu korrelieren, habe ich eine einfache und verlässige Methode entwickelt die es, basierend auf 2D Röntgen-videos niedriger Röntgendosis (~6,5mGy) erlaubt, in narkotisierten Mäusen die Lungenfunktion zu bewerten. Mit Hilfe dieser neuen Methode gelang es mir charakteristische Unterschiede in der Lungenfunktion von asthmatischen, therapierten und gesunden Mäusen in vivo über die Zeit nachzuweisen, und diese Resultate mit den Ergebnissen von CT und Histologie zu korrelieren. Das Verfahren wird derzeit von mir für die Anwendung an frei beweglichen und nicht narkotisierten Mäusen weiterentwickelt. Dies sollte zu einer deutlichen Stressreduktion für die Maus bei der Untersuchung führen und somit, vor allem in Asthma, im Gegensatz zu etablierten Verfahren wie Plethysmographie, die Erhebung validerer Messdaten erlauben. Mit Hilfe von „near infrared fluorescence imaging“ konnten wir in vivo und longitudinal erfolgreich verschiedene durch Asthma ausgelöste molekulare Veränderungen in der Mauslunge verfolgen. Erstens erlaubte die Verwendung einer neuen Polyglyzerol Probe mit dendritischer Struktur (MN2012) die spezifisch an Selektine bindet, die Darstellung der durch Asthma verursachten Entzündung der Lunge. Im Zuge dessen konnten wir nachweisen, dass sich MN2012  zur Darstellung von Enzymkinetiken bei Entzündungsreaktionen durch eine schnellere Kinetik und höher Spezifität als kommerziell erhältliche Proben auszeichnet. Zweitens haben wir gezeigt, dass in Kombination mit einem Fluoreszenz markiertem Antikörper gegen SiglecF, einem Antigen das hauptsächlich auf Eosinophilen exprimiert ist, Eosinophilie in asthmatischen Mäusen verfolgt und der Effekt einer Dexamethason Behandlung  ebenso dargestellt werden kann. Drittens konnten wir den Verbleib inhalierter fluoreszierender Nanopartikel in der Lunge der Maus in vivo untersuchen und dabei nachweisen, dass diese hauptsächlich von endogenen Makrophagen im Lungengewebe aufgenommen werden. Alle diese Techniken wurden gegeneinander und mittels histologischer Analyse und Fluoreszenzmikroskopie korreliert und validiert.  Zusammenfassend bilden die in meiner Dissertation entwickelten Lungenbildgebungsstrategien für Asthmamausmodelle eine Bildgebungsplattform, um sowohl spezifische Effekte in asthmatischen Mäusen unterschiedlichen Schweregrades als auch die Auswirkungen neuer Therapien abzubilden und im Detail zu untersuchen

Comparative analysis of squamate brains unveils multi-level variation in cerebellar architecture associated with locomotor specialization

Ecomorphological studies evaluating the impact of environmental and biological factors on the brain have so far focused on morphology or size measurements, and the ecological relevance of potential multi-level variations in brain architecture remains unclear in vertebrates. Here, we exploit the extraordinary ecomorphological diversity of squamates to assess brain phenotypic diversification with respect to locomotor specialization, by integrating single-cell distribution and transcriptomic data along with geometric morphometric, phylogenetic, and volumetric analysis of high-definition 3D models. We reveal significant changes in cerebellar shape and size as well as alternative spatial layouts of cortical neurons and dynamic gene expression that all correlate with locomotor behaviours. These findings show that locomotor mode is a strong predictor of cerebellar structure and pattern, suggesting that major behavioural transitions in squamates are evolutionarily correlated with mosaic brain changes. Furthermore, our study amplifies the concept of 'cerebrotype', initially proposed for vertebrate brain proportions, towards additional shape characters.Peer reviewe

In Pursuit of an Optimum Optical Imaging Technology for Early Detection of Dental Caries

Clinical caries detection techniques, such as radiographs, are not sensitive to detect and monitor the progression of caries at early stages. In recent years, several optics-based imaging modalities have been proposed for early detection of caries. In this thesis, we report on a systemic comparative study on the performances of optical coherence tomography and thermophotonic lock-in imaging (TPLI) as early caries detection imaging modalities based on light scattering and absorption, respectively. Through controlled demineralization on extracted human teeth, our results suggest that TPLI provides better sensitivity and detection threshold in detecting early stages of caries. The outcome justifies the need for a light-absorption based imaging modality to produce depth-resolved images. Therefore, preliminary imaging studies on a 3-D imaging platform known as a Truncated-Correlation Photothermal Coherence Tomography (TC-PCT) system was conducted to achieve optimal diagnostic yield. The results demonstrate that TC-PCT can detect early caries with significant enhancement in depth-resolution