高速及深組織光學顯微技術應用於果蠅腦功能之研究

High-speed and Deep-tissue Optical Microscope Techniques for Drosophila Brain Functional Studies

徐國仁(Kuo-Jen Hsu)

指導教授 ： 朱士維

國立臺灣大學/理學院/物理學研究所/博士(2018年)

https://doi.org/10.6342/NTU201803368

摘要

自從 Cajal 利用光學顯微鏡觀察神經組織開始，科學家已累積大量單一神經細 胞或是數個神經細胞的知識，但這仍不足讓我們全盤了解大腦的功能。大腦是由 數以千萬億微米等級的神經細胞形成的複雜三維網路，彼此間利用毫秒時間尺度 的動態行為運作。因此要了解大腦的功能，必須要發展適合的工具，利用非侵入 式的方式，在具有單一神經細胞的解析度下同時觀察活體全腦功能。
要研究活體全腦功能，傳統電生理學可量測毫秒解析度的神經活動，而功能 性腦磁振造影可非侵入式觀察人的全腦。但電生理學為侵入式觀察，且同時只能 觀察數量有限的細胞，而功能性腦磁振造影的解析度不足以觀察單一神經細胞， 且無法提供腦活動的直接結果。而光學方法可以以非侵入式觀察並提供單一神經 細胞解析度，且可以觀察小動物的全腦，是活體腦功能研究的最佳工具。本篇論 文中，由於果蠅完備的解剖腦圖譜，因此我們用果蠅做為研究對象。
利用光學方法研究活體腦功能時，常見的工具為共軛焦及雙光子顯微鏡，因 其具備光學切片能力，可應用於組織觀察。但切片限制了快速三維取像的速度， 本篇論文提出結合聲光變焦透鏡的商用雙光子顯微鏡的方法，提升三維取像速度， 它以數十萬至百萬赫茲的頻率做軸向的焦點掃描，掃描範圍達上百微米，結合掃 描系統的橫向任意曲線掃描，提供了一個三維光學緞帶成像的掃描方式。利用這 個新穎的成像模式可以觀察毫秒時間尺度的三維神經活動，且同時避免活體樣本 晃動造成的影響，對於高神經密度的果蠅腦是最佳的研究工具。
而在研究果蠅腦功能時，發現了另一個預料外的現象，就是雙光子顯微鏡並 無法穿透整個約兩百微米深的果蠅全腦，其主要原因為腦中氣管強烈的像差及散 射。為了讓光學影像能穿透果蠅全腦，本篇論文中利用了波長 1300 奈米的雷射， 搭配三光子螢光激發，首次實現了活體果蠅在亞細胞解析度下的全腦觀察。由於 長波長的光可以減少散射，並且同時降低波前誤差產生的像差，而三光子螢光提 供了相較於雙光子螢光更優異的光學切片能力。另外，我們也首次利用不同波長 的激發，定量的驗證並解釋限制雙光子影像在果蠅腦穿透深度的機制。在短波長激發下，散射是主要限制穿透深度的因素，然而，在長波長的激發下，像差的影 響超越了散射，成為主要限制影像穿透深度的原因。
藉由以上高速及深組織的光學顯微技術開發，以及全面性的了解果蠅腦的光 學特性，完成果蠅功能腦圖譜的目標指日可待。

關鍵字

多光子顯微鏡 ； 快速三維光學緞帶成像 ； 功能型腦圖譜 ； 全腦觀察 ； 訊號衰減 ； 綠螢光蛋白

並列摘要

Since the day of Cajal, neuroscientists have accumulated significant amount of knowledge of single neuron or few-neuron circuits. However, to understand the emergent properties of brain, which composed of three-dimensional (3D) networks from thousands to millions of micron-sized neurons with millisecond to second temporal dynamics, suitable tools should be adopted to explore the functional dynamics throughout whole living brain with single neuron spatiotemporal resolution, i.e., functional connectome.
To study functional connectome, electrophysiology has been successfully applied to single neuron measurements with millisecond resolution in an intact brain, and functional magnetic resonance imaging (fMRI) has been widely used to study whole human brain functional properties. However, electrophysiology is invasive, and the number of simultaneously monitored neurons is limited, while fMRI provides only indirect results of brain activities and nonsufficient spatiotemporal resolution to distinguish single neuron. On the other hand, optical methods provide noninvasive measurements, high spatiotemporal resolution to distinguish single neuron, and whole-brain observation when applied to small animal brains, is the optimal tool. In this dissertation, Drosophila is selected as our research target due to its nearly-complete anatomical connectome.
When using optical method to study the brain, confocal/two-photon microscope (2PM) is widely adopted due to their sectioning capability, which is suitable for tissue inspection. However, their 3D acquisition speeds are limited due to sectioning. In this dissertation, we enhance 3D acquisition speed by integrating an ultrasound lens (UL) with a commercial 2PM, providing hundreds of kHz to one MHz axial scan rate with more than 100 μm axial extent. Combined with a commercial scanner that allows arbitrary curve scan on lateral plane, a novel ribbon scan imaging modality is developed. It is demonstrated to monitor millisecond temporal dynamics of 3D neurons of interest without motion artifacts, which is best suited for densely-packed Drosophila brain.
During Drosophila brain functional studies, it is unexpectedly discovered that 2PM cannot penetrated the whole ~ 200 μm living brain. The reason is the extraordinary strong aberration/scattering from the tracheae structures. To improve imaging depth, a 1300-nm laser combined with three-photon excitation (3PE) is adopted to achieve whole-brain observation with subcellular resolution for the first time. The long excitation wavelength simultaneously reduces scattering, and aberration caused by phase error. In addition, 3PE process renders exceptional optical section capability. To explore the mechanism that limit two-photon imaging depth in Drosophila brains, the brain optical properties at various excitation wavelengths are quantitatively characterized for the first time. Surprisingly, at short wavelength, scattering dominates; while aberration exceeds it at long wavelengths and becomes the main impeding factor of whole-brain observation in a living Drosophila.
Through the validations of the 3D high-speed and deep-tissue optical imaging techniques, together with comprehensive understanding of light interaction in Drosophila brains, it paves the way toward constructing the first whole-Drosophila-brain functional connectome.

並列關鍵字

multiphoton microscopy ； fast 3D optical ribbon imaging ； functional connectome ； whole-brain observation ； signal attenuations ； green-fluorescence protein

參考文獻

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