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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 朱士維(Shi-Wei Chu) | |
dc.contributor.author | Kai-Ping Yang | en |
dc.contributor.author | 楊凱評 | zh_TW |
dc.date.accessioned | 2021-05-19T17:54:18Z | - |
dc.date.available | 2022-02-20 | |
dc.date.available | 2021-05-19T17:54:18Z | - |
dc.date.copyright | 2017-02-20 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2017-02-14 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7808 | - |
dc.description.abstract | 大腦是掌管動物一切行為、情緒、意識、生命徵象等活動的重要器官,過去數十年來,神經科學研究有許多進展,例如發現許多神經疾病與大腦功能的關聯性。科學家們致力於了解腦神經科學網絡,使得腦神經細胞間的訊息傳遞逐漸成為重要且熱門的研究主題,研究大腦功能需要適當工具刺激與記錄神經訊號,但由於神經細胞體積較小、訊號傳遞迅速,且大腦又是相當緊密複雜的立體結構,因此,若要進行活體實驗,則需要發展具有高時間/空間解析度,能快速記錄立體結構中訊息傳遞的非侵入性技術。
傳統上,神經實驗多以電生理學方式進行,利用微電極刺激與記錄細胞膜電位變化來研究神經訊號傳遞,但此種方法不僅具有侵入性,也因為尺寸大小限制使得電極數量有其極限,再者,要將兩根微電極插入極靠近的神經元中以提高解析度也相當困難。相較之下,全光學式生理學以光學方式激發與記錄神經訊號傳遞,不僅提供了一種非侵入式的觀測方法,且可在同時偵測多個神經元時仍保持高空間解析度,十分有利於觀測活體中的神經網絡訊號傳遞。 然而,由於影像擷取速度較慢,現有的全光學式生理學仍然無法提供在三維空間中毫秒等級的空間解析度,因此,本研究的主要貢獻在於結合全光學式生理學與高速三維影像技術以改善現有顯微技術。由於活體內的腦神經元活動多半分布於立體空間中,若要觀察網絡中的訊息傳遞,勢必需要三維空間的影像顯微技術,且神經訊號傳遞快速,故此影像擷取技術也須於短時間內取得分布於三維空間中的訊號變化。在本研究中,我們在系統內加入可調式聲光折射率梯度透鏡,提供高速軸向變焦能力,可大幅降低取得三維影像訊號所需之掃描時間,且受到樣本空間穩定性的限制較小,使系統能更有效記錄三維空間中的神經訊號傳遞。 另一方面,在樣本的選擇上,果蠅是相關研究中常用的模式生物。由於果蠅腦與人腦結構有高度相似性,生命週期短、子代數量龐大,神經網絡複雜度又低於人腦,且相關基因轉殖技術也較為成熟,故相當適合作為研究樣本。在實驗中,我們使用基因轉殖的果蠅作為樣本,將紅光光敏離子通道蛋白(ReaChR、CsChrimson)及鈣離子濃度指示劑綠螢光蛋白(GCaMP6f)表現在果蠅的上下游目標腦區,當光敏通道蛋白受到單光子紅光(638 nm)雷射激發時,離子通道會開啟使上游神經細胞去極化產生動作電位,此激發方式比微電極更精準也更有彈性。而當訊號傳至下游腦區時,動作電位會導致細胞間鈣離子的流動,綠螢光蛋白會在鈣離子濃度改變時產生亮度變化,此亮度變化可被雙光子顯微鏡記錄下來,從而將神經細胞的動作電位訊號視覺化,使用此兩種基因轉殖技術即可以全光學的方式完成神經訊號的激發與記錄。 綜上所述,全光學式生理學結合三維影像顯微技術提供了非侵入性、時間/空間解析度高及三維影像掃描時間短等優勢,讓科學家能用光學基因轉殖激發、高速三維光學紀錄的方式了解神經細胞訊號傳遞,有助於釐清腦神經網絡中的上下游關係,進而更全面地理解大腦的運作方式,是腦神經科學研究領域中強而有力的工具。 | zh_TW |
dc.description.abstract | Brain, which controls behaviors, emotions, consciousness and all vital signs of animals, is one of the most important organs in body. In recent decades, there have been significant progresses in neuroscience, such as the discovery of relations between some neural diseases and specific brain functions from the Human Connectome Project in USA since 2009. Scientists are dedicated to study the way how connectome works in brain, in order to provide new mechanistic insights of how brain functions affects memories, behaviors and diseases, thus made connectome a significant and popular topic nowadays. To understand the functional connectome of brain, we need to start from studying neural signaling. However, due to the small size (~μm) and the fast response (~ms) of neuron signaling, and the complex 3D structures of brain for in vivo experiments, it is necessary to develop suitable tools that have high spatial/temporal resolutions, fast volumetric recording ability and non-invasive feature.
The conventional way is electrophysiology, which uses microelectrodes to stimulate and record electrical signals across neural membranes. However, this method is invasive, and spatially limited to one or few selected neurons due to the size of electrodes. Moreover, it is difficult to achieve high spatial resolution with two nearby microelectrodes inserted closely in neuron cells. In contrast, all-optical physiology, which uses light to both stimulate and record neural signaling, not only provides a noninvasive experiment method, but also achieves high spatial resolution when imaging tens to hundreds of neurons simultaneously. Nevertheless, due to the slow acquisition speed of imaging, current all-optical physiology studies have not achieved millisecond-scale temporal resolution in 3D tissue. The main novelty of our work is to improve the existed microscope by combining all-optical physiology with fast volumetric imaging technique. For monitoring in vivo fast neuronal activities in three-dimensional space, it is necessary to adopt a high-speed volumetric imaging microscopy to get the signal variation within very short time. In this research, we inserted a tunable acoustic gradient-index (TAG) lens into the all-optical physiology microscope system to rapidly change the focus position of recording laser in axial direction. It can significantly reduce the acquisition time of 3D imaging with less requirement of spatial stability of sample, therefore is more efficient in observing neural dynamics. On the other hand, in terms of model animal selection, Drosophila, which has short lifespan, large number of offspring and various mutations, is feasible to be genetically encoded and statistically analyzed. Since Drosophila brain has similar functional features to human brain, but with less complexity, it is a suitable model organism in brain research. In this work, we chose genetically modified Drosophila as the model sample. The Drosophila was encoded with ReaChR or CsChrimson actuators in a specific brain region, and was labeled with GCaMP6f sensors in a downstream region. When the ReaChR or CsChrimson is activated by 1-photon stimulation with a red laser (638 nm), the ion channels open and depolarize the neurons to produce action potentials. Following that, when the signals go to a downstream region, the action potential will induce the release of calcium ions, and then the GCaMP6f will show fluorescence intensity variation related to the concentration of calcium ions. This variation of fluorescence intensity can be recorded by a 2-photon microscope, enabling the visualization of action potential. That is to say, these optogenetic actuators and sensors help achieving optical stimulation and observation. In conclusion, all-optical physiology combining with volumetric imaging technique provides the advantages of noninvasiveness, high spatiotemporal resolution and fast 3D scanning. By simultaneously using optogenetic stimulation and high-speed volumetric observation, we are capable to study neuron signal propagations and sequences in live connectome. This technique is a powerful and innovative tool in neuroscience research. | en |
dc.description.provenance | Made available in DSpace on 2021-05-19T17:54:18Z (GMT). No. of bitstreams: 1 ntu-105-R03222016-1.pdf: 4151263 bytes, checksum: 88e98bbc96955c1c35d14abcdda7e093 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員會審定書 #
誌謝 i 中文摘要 iii ABSTRACT v CONTENTS viii LIST OF FIGURES xi LIST OF TABLES xiii Chapter 1 Introduction 1 1.1 Importance and Difficulties of Brain Research 1 1.2 Advantages of Drosophila Sample 1 1.3 Advantages of Optical Stimulating and Recording 2 1.4 Aim and Structure of This Dissertation 4 Chapter 2 Recording and Stimulating Neural Signals 5 2.1 Neuron Signaling 5 2.2 Recording and Stimulating 7 2.2.1 Recording Methods: Electrical, Magnetic, and Optical Methods 7 2.2.2 Stimulating Methods: Electrical, Chemical, Sensory, and Optical Methods 10 Chapter 3 All-Optical Physiology: Recording and Stimulating 16 3.1 Optical Recording Methods 16 3.1.1 Optogenetic Sensors: Genetically Encoded Calcium Indicators (GECIs) 16 3.1.2 Deep In Vivo Imaging: Multiphoton Microscopy 18 3.1.3 Fast Volumetric Imaging 19 3.2 Optical Stimulating Methods 26 3.2.1 Optogenetic Actuators: Genetically Encoded Opsins 26 3.2.2 1-Photon Stimulation vs 2-Photon Stimulation 27 3.3 Simultaneous Stimulating and Recording: Single-Scanner System vs Dual-Scanner System 28 Chapter 4 Experiment Setup for All-Optical, Volumetric Physiology 31 4.1 Hardware 31 4.2 Software 36 Chapter 5 Samples 38 5.1 Drosophila Melanogaster: Preparation 38 5.2 Selection of GECI: GCaMP6f 39 5.3 Selection of Opsins: ReaChR & CsChrimson 39 5.4 Selection of Systems in Brain: Olfactory System & Visual System 41 Chapter 6 Experiments Methods (Supplementary) 45 6.1 System Setting 45 6.2 Stimulation Laser Calibration and Test 46 6.3 Sample Preparation and Dissection 49 6.4 Volumetric Imaging 50 6.5 All-Optical Physiology Functional Imaging 53 Chapter 7 Results and Discussions 55 7.1 Stimulation Laser Calibration Test 55 7.1.1 Results 55 7.1.2 Discussion 56 7.2 Volumetric Imaging Test 57 7.2.1 Results 57 7.2.2 Discussion 64 7.3 Initial Functional Imaging Test of ReaChR & GCaMP6f in Olfactory System (Failed) 65 7.3.1 Results 65 7.3.2 Discussion 69 7.4 Second Functional Imaging Test of CsChrimson & GCaMP6f in Visual System 69 7.4.1 Results 69 7.4.2 Discussion 72 7.5 Dual-Scanner System Test 72 7.5.1 Results 72 7.5.2 Discussion 78 7.6 All-Optical Physiology and Volumetric Functional Imaging 80 7.6.1 Results 80 7.6.2 Discussion 84 Chapter 8 Conclusions and Prospections 86 8.1 Conclusions 86 8.2 Prospections 87 REFERENCE 90 | |
dc.language.iso | en | |
dc.title | 全光學式生理學與三維影像顯微技術於果蠅神經研究之應用 | zh_TW |
dc.title | The Application of All-Optical Physiology and Volumetric Imaging Microscopy in Drosophila Neuroscience Research | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳摘文(Tsai-Wen Chen),江安世(Ann-Shyn Chiang),林彥穎(Yen-Yin Lin) | |
dc.subject.keyword | 全光學式生理學,三維影像,變焦透鏡,光學顯微鏡,光遺傳學,果蠅腦神經網絡, | zh_TW |
dc.subject.keyword | All-Optical Physiology,Volumetric Imaging,Tunable Lens,Optical Microscopy,Optogenetics,Drosophila Connectome, | en |
dc.relation.page | 97 | |
dc.identifier.doi | 10.6342/NTU201700566 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2017-02-14 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 物理學研究所 | zh_TW |
顯示於系所單位: | 物理學系 |
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ntu-105-1.pdf | 4.05 MB | Adobe PDF | 檢視/開啟 |
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