以共軛干涉式散射顯微鏡偵測腦組織內高速動態訊號

Abstract

大腦掌控著動物的日常功能、情緒、意識、生命徵象等的重要器官。為了瞭解大腦複雜的功能運作，我們需要從最小單元出發，以神經元訊號的產生以及傳遞來切入，再進一步理解神經間功能性訊號的溝通。透過不同技術可以觀測到這些訊號的發生，如電生理學量測神經細胞的電訊號變化、螢光影像觀察特定離子訊號強度變化等，在這些眾多方法之中無標記影像技術提供了低侵入性、高時間/空間解析度以及可以避免標記式影像技術所導致的缺點，如螢光蛋白的光漂白、標記所需的成本及對樣品的影響。 神經訊號來源於動作電位的產生以及神經之間的離子流動，這些訊號具有訊號微弱、發生時間尺度小的特性造成觀測上的困難，加上神經訊號傳遞的過程建立於組織層級上，具有足夠的立體空間解析度也是另一個挑戰。因此我們透過開發具有高靈敏度、高速且具有軸向解析度的無標記影像技術以提供對於神經訊號新的觀察方式，也同時提供了之前技術所不具有的優點和更多資訊。 在使用無標記影像取得大腦功能性訊號的研究中，干涉式影像技術通過其對相位敏感的優點，尤其適合被用來偵測腦中這些微小訊號。干涉式散射顯微鏡(iSCAT)在干涉式影像技術之中擁有非常優異的靈敏度，可有效地測量這些訊號的變化，而普遍的干涉式散射顯微鏡不具有光學切片的能力，因此為了在腦組織的三維空間中偵測靈敏且快速的訊號變化，我們結合旋轉盤單元組件與干涉式散射顯微鏡開發了共軛焦干涉式散射顯微鏡 (C-iSCAT)，使其具有光學切片和高速擷取的能力，加上客製元件達成光偏振方向的控制以及軟體調整，我們成功建構出具有高靈敏度的光學影像系統。另外在技術開發階段中，利用不同樣本進一步確認顯微鏡靈敏度、對比以及解析度，確認在多數情形下我們都能透過此系統取得具有足夠靈敏度且高速的影像訊號。 在本研究中，我們透過相對簡單的樣品如奈米粒子和生物培養細胞來展示我們系統的性能表現，其能夠穩定的在高速攝影（1000 fps) 下拍攝10奈米金粒子以及捕捉細胞內胞器如粒線體與內質網的動態影像。在腦成像方面，我們觀察到果蠅腦內的結構信息，可以觀察特定腦區如蕈狀體(MB)及觸角葉(AL)呈現不同的對比度，同時我們對動態的干涉散射訊號進行時間上變異性分析顯示腦內活動與訊號波動之間的關係。為了進一步獲取與刺激同步的功能訊號，我們利用光遺傳學技術在特定區域標記ChR2基因的果蠅樣本下進行藍光刺激實驗，通過傅立葉分析得到頻譜上的資訊，另外也試著透過統計不同實驗數據間時間上變異性的大小比較來分析刺激所導致的訊號變化。

The brain plays a crucial role in controlling various physiological functions, emotions, consciousness, and vital signs in animals. To gain a comprehensive understanding of its complex workings, it is essential to study and record neuronal signals. Neuronal signaling occurs through the generation of action potentials and the transmission of ions influx. In recent years, several techniques have been developed for studying and recording these signals. Among them, label-free optical imaging approaches offer advantages such as reduced invasiveness, high spatiotemporal resolution, and the avoidance of labeling limitations, such as photobleaching, the cost for labeling and its potential effects. In the field of label-free imaging for functional signal study, interferometric imaging techniques enable phase-sensitive detection of weak signals. While these interference-based approaches provide quantitative phase retrieval or phase information, interference reflection microscopy excels in sensitivity, which is critical for capturing the weak scattering signals associated with brain activity. Previous related studies mainly focused on the single neuron scale instead of brain tissue. To effectively capture these signals in the brain and detect the subtle and rapid signal changes in three-dimensional distribution, we developed confocal interference microscopy (CIM) by combining it with a spinning-disk unit. This combination allows for optical sectioning and fast acquisition speed. Moreover, through the design of polarization maintaining with customized elements and the system synchronization, we successfully developed the optical imaging system with high sensitivity. We carefully considered the principle in various conditions during observations with our system and described how it primarily functions as a phase-sensitive microscope. In this work, we first demonstrated the system's performance in nanoparticles and cell samples, with the observation of the cellular organelles dynamics and the recording of 10 nm AuNPs under 1000 fps. We were able to observe clear dynamics of intracellular organelles. In brain imaging, we utilized our system to capture both in vivo and ex vivo Drosophila samples, generating structural contrast images. We also conducted an analysis of temporal fluctuations in the interferometric scattering signals, comparing different conditions. To detect brain functional signals, we designed a stimulation experiment using genetically modified Drosophila samples expressing ChR2 in specific brain regions. We recorded the interferometric scattering signals while activating ChR2 through stimulation at specific frequencies using a blue laser (488nm). We further applied Fourier transform analysis to examine the frequency components of the signals and conducted statistical analyses on the temporal variation differences during stimulation across multiple conditions. Unfortunately, we were unable to extract clear functional signals from the complex interferometric signals. However, there are still potential avenues for exploration in future studies. We provide further suggestions for the project's future directions and improvements.