Title

以20奈米解析度觀察果蠅全腦神經網路

Translated Titles

Observing neuronal network across the whole brain of Drosophila with ~20 nm resolution

DOI

10.6342/NTU201900735

Authors

林涵源

Key Words

定位顯微術 ; 轉盤共軛焦顯微鏡 ; 光學澄清 ; 深組織中的超解析顯微術 ; 神經網路 ; localization microscopy ; dSTORM ; spinning disk confocal microscope ; tissue clearing ; deep-tissue superresolution ; neuronal network

PublicationName

臺灣大學物理學研究所學位論文

Volume or Term/Year and Month of Publication

2019年

Academic Degree Category

碩士

Advisor

朱士維

Content Language

英文

Chinese Abstract

了解神經網絡的分布有助於知道在腦中的神經如何連結、甚至如何運作的,但是神經纖維相當細緻,又會彼此纏繞形成複雜結構,用傳統顯微鏡非常不容易分辨出來。比方說,神經的樹突纖維可以小到100奈米的寬度,而當兩條纖維彼此緊靠,我們需要20奈米的解析度才能分辨(假設細胞膜厚度10奈米,螢光表現在細胞質)。更困難的地方是這些細緻的神經纖維會在三度空間的腦組織中延伸很遠,連結到遠方的神經。所以若想要清楚的分析神經網路,我們不只需要高解析度,還需要這個解析度能夠維持穿透整個腦的深度。 因為繞射的限制,光學系統的最佳解析度大約為激發波長的一半,以可見光來說就是約250奈米。2014年的諾貝爾化學獎頒給了三位科學家,他們致力於發展可以突破繞射極限的超解析顯微術。其中,定位顯微術只需要接收到100個光子,就可以把螢光分子的位置定位到接近20奈米。因此,若需要20奈米解析度,螢光定位顯微術會是最好的選擇。 但要在腦中使用定位顯微術會遭遇到三個困難。第一是對比,定位顯微術需要能夠不斷閃爍的對比劑,而基因轉殖的螢光蛋白比較容易標記整個腦組織,但是普通的螢光蛋白只會發出強度固定的螢光,並不會閃爍,而且存在著光漂白(Photo-bleach)的問題;第二是廣域照射(Wide-field)的定位顯微術缺乏光切片(Optical sectioning)的能力,沒辦法區辨不同層的訊號,所以一般定位顯微術被侷限在數個微米的深度內;第三是來自組織的像差、散射也同樣會限制影像深度。 因此,我們結合四個關鍵的技術:一、以定位顯微術提升解析度;二、使用還原劑(ME)來使基因轉殖、可以光轉換的螢光蛋白閃爍,並在其被光漂白後,利用光轉換來補充螢光蛋白;三、利用轉盤共軛焦顯微鏡提供光切片的能力;四、使用光學澄清減少組織的散射與像差。藉此,在果蠅腦從上到下的任何位置都取得20奈米超解析影像。而且藉由這個方法,我們可以解決一開始提出的問題:區辨兩團靠得很近的樹突纖維,甚至以三維重組呈現它在空間中的走向。

English Abstract

To understand brain function, detailed anatomical mapping of neurons and their fiber distributions should be the first step. However, it is not an easy task since neuronal network is composed of tiny fibers that may closely entangle with each other. For example, the diameter of a dendrite in Drosophila brain can be as small as 100 nm, and when dendritic fibers interweave with each other, at least 20-nm resolution would be necessary to resolve these fibers (assuming fluorescent protein expressed in cytoplasm, and the thickness of a cellular membrane is 10 nm). What makes it even more difficult is that these fibers may extend three dimensionally throughout a brain, so we need not only a high-resolution technique, but also a technique that is able to penetrate the whole brain to track neuronal fibers. Because of diffraction limit, the resolution of an optical microscope is confined around λ/2, which is roughly equal to 250 nm for visible light. Nobel Prize in Chemistry 2014 was awarded to three scientists due to their contribution on “superresolution microscopy” that is able to break the diffraction barrier. Among all superresolution techniques, localization microscopy achieves one order resolution enhancement by detection of >100 photons from each fluorescent protein (FP). Thus, to reach 20-nm resolution, localization microscopy should be the best option. Nevertheless, to apply localization microscopy across a whole brain, there are three major challenges. First, localization microscopy requires a “blinking” contrast agent. Generally speaking, genetically encoded FP is used when labeling a thick brain tissue. However, blinking FP is scarce, while suffered from photo-bleaching. Second, due to lack of optical sectioning ability, wide-field-based localization microscopy cannot distinguish signals from different layers and thus imaging depth is confined to less than 10 μm. Third, tissue-induced scattering and aberration also limit the imaging depth. In this study, we combine four techniques to achieve 20-nm superresolution across a whole Drosophila brain, including 1. localization microscopy to enhance resolution; 2. adding ME to enable blinking of genetically encoded, photo-convertible FP. Furthermore, we can use photo-conversion to supply the bleached FP; 3. spinning disk confocal microscope to provide optical sectioning; 4. tissue clearing to minimize tissue scattering/aberration. We name this combination COOL, i.e. spinning disk COnfocal lOcalization with tissue cLearing. COOL allows us to distinguish 3D entangled dendrites even at the bottom of the brain, paving the way toward whole-brain neural network analysis.

Topic Category 基礎與應用科學 > 物理
理學院 > 物理學研究所
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