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標題: | 全光學式生理學與快速三維影像顯微技術於果蠅腦神經功能性連結研究之應用 All-Optical Physiology with Fast Volumetric Imaging Microscopy Applied in Drosophila Brain Neuron Functional Connection Study |
作者: | Chiao Huang 黃喬 |
指導教授: | 朱士維(Shi-Wei Chu) |
關鍵字: | 全光學式生理學,雙光子顯微術,高速影像擷取,可變焦透鏡,腦神經功能性連結網絡, All-optical physiology,two-photon microscopy,high-speed imaging,focus tunable lens,brain functional connectome, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 大腦的運作由其複雜的腦神經網絡聯結所掌控。雖然藉由電子、光學顯微鏡,科學家可以解析出結構上相連接的神經,但想要全面性地解讀神經網絡的功能,我們仍然需要瞭解神經之間功能上的聯結特性,包括反應時間順序、類型以及強度等資訊。
神經藉由傳遞電訊號作為溝通的橋樑,因此,透過刺激上游神經,並記錄下游神經的反應,我們可以瞭解神經群之間的功能性聯結。由於神經細胞空間尺度在微米等級、訊號傳遞的時間也在毫秒尺度,我們必需使用高空間/時間解析度的技術來進行功能性聯結的研究。 傳統上,科學家會藉由電生理的方法,以微米大小的電極對神經進行刺激和記錄。這種方法提供了相當優異的訊噪比及微秒等級時間解析度,然而,這種方法不但具有侵入性,也不適用於在空間上密集分布的神經群。相較之下,全光學式生理學以光對神經進行激發和紀錄,除了具有非侵入性的優勢,也提供了次微米/毫秒等級的空間/時間解析,因此非常適合用於觀察活體的神經網路聯結。 雖然過去已發展出許多全光學式生理學 ── 包含刺激和紀錄的技術,但記錄端幾乎都只限於單一深度的影像擷取。由於神經間的聯結分布於三維空間,其中的訊號傳遞十分快速,因此,勢必需要將全光學式生理學與快速三維影像顯微技術結合。在本篇論文中,藉由結合可調式聲光折射率梯度透鏡,我們建立了雙光子快速體積紀錄系統,並與單光子點刺激系統結合,提供了一個可進行三維空間全光學式生理學研究的平台。 此平台以應用於探討活體果蠅視覺神經路徑(anterior visual pathway, MED→AOTU→BU) 的功能性聯結作為例子,藉由刺激連接MED與AOTU單一單元區域(AOTUil)內的上游神經,與觀察連接AOTU與BU之下游神經的反應,建構出三維空間中AOTUil與BU中密集分佈的單元結構 (microglomeruli) 之功能性聯結圖譜。 綜上所述,此全光學式生理學平台提供了非侵入性、精準刺激,及高時間/空間解析度體積紀錄的優勢,對於研究活體腦神經功能性聯結而言,是相當強而有力的新工具。 How the brain works is based on its complex connection map in neuron networks, i.e. connectome. Although structural connection among neurons can be unraveled by anatomical analysis, to fully understand the function of the circuit, it is essential to investigate their functional connection properties, including temporal sequence, type, as well as interaction strength of the connections Function of neuron circuit is based on the electrical signals transmitted among interconnected neurons. To investigate this, it is critical to stimulate upstream neurons and recording their downstream counterparts. Due to the small size (~μm) and fast response (ms~sec) of the neurons, stimulation/recording tools with high spatiotemporal resolution are critical. Electrophysiology, which stimulates and records neuron signal through microelectrodes, provides micron and microsecond spatiotemporal resolution with high signal to noise ratio, and have long been used as a golden standard for neuron functional connection study. However, it is an invasive method and it is hard to interrogate multiple densely packed neurons. In contrast, all-optical physiology, which uses light to stimulate and record neurons, is an emerging tool allowing study of multiple neurons non-invasively with submicron and millisecond spatiotemporal resolution. Although several all-optical physiology platforms, including stimulation and recording techniques, have been demonstrated, the recording designs are mostly limited to single-depth imaging. Due to 3D distribution and fast responses of neurons, integrating high-speed volumetric imaging in all-optical physiology setup is highly desired. In this thesis, by using a tunable acoustic gradient-index lens, which is a fast axial scanning device, two-photon 3D recording is achieved. Combining with single-photon stimulation, a three-dimensional all-optical physiology platform is constructed. The usefulness of the platform is demonstrated on in-vivo functional connection study of Drosophila anterior visual pathway (MED→AOTU→BU). By stimulation on upstream neurons (MT neurons, MED→AOTU) in a single sub-unit of AOTU (AOTUil), the responses in corresponding downstream neurons (TB neurons, AOTU→BU) can be fully observed in 3D, unraveling the functional connection coding between AOTU and tens of BU microglomeruli, which are tiny subunits with 2-3 micron diameter and densely packed in ~ 35 μm × 35 μm × 40 μm volume. In summary, with high spatiotemporal resolution volumetric recording and precise stimulation, this work paves the way toward non-invasive investigation on 3D brain functional connectome in-vivo. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77509 |
DOI: | 10.6342/NTU201803442 |
全文授權: | 未授權 |
顯示於系所單位: | 應用物理研究所 |
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