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標題: | 多焦雙光子體積顯微鏡於果蠅腦之功能性影像 Functional Volumetric Imaging of Drosophila and System Improvement of High-speed Multifocal Two-photon Microscopy |
作者: | Po-Yuan Wang 汪伯元 |
指導教授: | 朱士維(Shi-Wei Chu) 朱士維(Shi-Wei Chu | swchu@phys.ntu.edu.tw | ), |
關鍵字: | 多焦點顯微鏡,雙光子顯微鏡,可調式聲波梯度折射率透鏡,體積成像,功能性影像, Multifocal microscopy,Two-photon microscopy,Tunable acoustic gradient-index lens,Volumetric imaging,Functional imaging, |
出版年 : | 2022 |
學位: | 碩士 |
摘要: | 為了了解大腦的機制,腦神經影像技術在一個多世紀以來經歷了許多突破性的發展,然而我們對大腦的了解依然有限。迄今為止仍然沒有理想的影像技術能夠同時在整個大腦中實現足夠高的時空間解析度。為了達成這項目標,此研究裡我們以果蠅作為我們的模式生物,因為其極小的大腦體積,且具有相對完整的神經結構性圖譜。為了取得活體果蠅全腦功能性影像,我們需要達到以下需求:(I)非侵入性觀測;(II)微米尺度的空間解析度以分辨單一神經元;(III)~一百微米的穿透深度以進行果蠅腦中的深組織影像;(IV)毫秒等級時間解析度的體積影像以觀察三維空間中的功能性影像。 由於雙光子光學顯微鏡的眾多優點,其經常被用於大腦功能性研究,例如其非侵入性及微米尺度的空間解析度,光學切片能力亦適合進行深組織影像,達成了需求(I~III)。然而,大部分雙光子顯微鏡透過掃描整個樣本以獲得三維影像,限制了其影像速度。在此研究中我們分別在橫向及軸向加快成像速度以達成高速三維影像。在橫向上,我們利用繞射分光元件將單道光分成32道光,並使用32通道的光電倍增管提高其影像速度。在軸向上,我們使用可調式聲波梯度折射率透鏡達成焦點在此軸上的高速掃描。透過這結合這兩項技術,我們實現了時間解析度為兩毫秒的體積影像,即達成需求(IV)。 此研究使用鈣離子螢光蛋白(GCaMP7f)標記的果蠅腦進行活體觀測,透過觀察自發活動,發現了果蠅腦內蕈狀體中不同亞區的瞬時反應。通過進一步以電擊刺激果蠅,我們在頻域中觀察到週期性活動。證明了此系統能夠觀察活體果蠅腦的功能性影像。然而,在我們系統中還有一個需要被解決的問題:資料擷取過程中的數據遺失。可能的原因為軟體的過度負載,造成中央處理器的低處理效率。在未來更換較高效能的中央處理器後,期望我們所建立的高速多焦點多光子體積顯微鏡,能夠對於未來建立果蠅大腦的功能性神經連接圖譜有所幫助。 Brain is an important organ that plays a necessary role in our emotions, thought, memory, and almost every process that regulates our body. In order to understand the mechanism of the brain, numerous studies have progressed for more than a century. However, our understanding of the brain is still limited. The reason is that no ideal imaging tool nowadays has the capability to simultaneously achieve micrometer and millisecond spatiotemporal resolution in the whole brain. To achieve this requirement, we select Drosophila to be our sample because of its small brain size and the nearly-complete structural connectome. In order to accomplish in vivo whole Drosophila brain functional imaging, we need to reach the following requirements: (I) noninvasive method, (II) micrometer spatial resolution to distinguish neurons, (III) ~100 μm penetration depth for Drosophila deep tissue imaging, (IV) millisecond temporal resolution volumetric imaging for 3D functional dynamics. Two-photon microscopy (2PM) is often used for in vivo brain study because of its noninvasive characteristic, ~μm scale spatial resolution and optical sectioning that offers remarkable penetration depth, achieving the requirements (I - III), respectively. However, the imaging speed is limited since 2PM typically requires raster scanning through the whole sample. Here we increase the speed on the lateral axis via multifocal imaging formed by a diffractive optical element (DOE) and a multichannel PMT. The axial speed is enhanced by a tunable acoustic gradient-index (TAG) lens, which scans the focus on the axial axis with an ~100 kHz. Through combining these two optical elements, we are able to achieve volumetric imaging with ~2 ms temporal resolution, which accomplishes the last requirement (IV). In this study, we apply our system on the GCaMP7f-labeled Drosophila brains for in vivo imaging. With spontaneous activities, we discover the distinct transient response in different subcompartments in the mushroom body. We further stimulate the Drosophila via electric shock, and observe periodic activities in the frequency domain. However, one challenge remains is the data lost during imaging, which is caused by the software-induced low CPU processing efficiency. With replacing a high-performance CPU to overcome this difficulty in the future, our high-speed multifocal multiphoton volumetric microscope paves the way toward establishing functional connectome in Drosophila brain. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/86528 |
DOI: | 10.6342/NTU202204017 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2022-09-30 |
顯示於系所單位: | 物理學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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U0001-2509202221061500.pdf | 7.14 MB | Adobe PDF | 檢視/開啟 |
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