腦功能影像用微型化雙光子顯微鏡系統開發

Abstract

解密及揭開腦神經功能性影像一直是這個世紀最重要的科學議題之一，因此於此篇論文中，會發表一項全新開發的雙光子微型化內視鏡技術，不只可以用來知悉小鼠中腦區 (黑質緻密部腦區)(深度:4.3 mm)的功能性神經影像並且可以看到清楚的神經脊隨柱，對於腦神經科學在功能性影像上是非常重要的議題。

因此，我們開發出一套雙光子螢光微型化顯微內視鏡系統，用於在長時間尺度的觀測小鼠深腦區的細胞活動影像。由於是使用點掃描式的雙光子影像系統，又需要減輕整體顯微鏡的重量，因此挑選微機電掃描鏡(MEMS)及搭配漸變折射柱狀透鏡(GRIN lens)，以看到更深層的腦區。於此篇論文中，主要分為三個部分進行詳細說明，包含光學設計使用Zemax軟體模擬出2P的理論解析度為1.14um，視野範圍為123 〖μm〗^2、光機械結構設計使用Shapr3D軟體並使用3D列印機器進行列印、發展獨特對光技巧(籠式洞口對準技術)等的開發適用於此微型化系統、C++/ CLI 圖形化使用介面的開發，用於做高速資料擷取以及使用圖形化介面卡做即時影像高速處理。

然而，若要看到細胞的神經脊隨柱影像，那麼此顯微鏡的解析度必須要為微米等級的橫向解析度。以及由於希望未來能在小鼠活動時，觀測其腦中的神經功能性影像，因此此套顯微鏡系統必須是基於光纖來傳輸激發的雷射光，以及收螢光訊號。順道一提，在做活體實驗之前，要先將病毒(AAV9-syn-GCamp6f)打到我們有興趣的腦區，以有利於我們影像螢光的收集及觀測。

在此第一代NTU miniScope的開發下，主要分為兩個部分，分別為「影像模組」、「底座模組」，兩者是用磁吸式的方式做定位跟固定，是可以做拆裝的設計。也根據我們所做的光學設計，可以根據我們今日想要觀察的腦區，只要替換「底座模組」即可! 總結來說，此創新的NTU miniScope的設計，其重量為5克、尺寸為22mm x 6 mm x 21 mm、影像幀率為20 frames/sec、橫向解析度可以達到1.292 ± 0.012 微米、視野範圍為128{\\mu m}^2。並搭配著特殊的光機械設計，可以看到黑質緻密部腦區的神經脊隨柱功能性影像。

Decoding and unveiling the neuron functions have become one of the most significant scientific challenges of this century. In this paper, we present a newly developed two-photon miniaturized endoscopic technology, which not only allows for the visualization of functional neural activities in the midbrain region (specifically the substantia nigra pars compacta, at a depth of 4.3 mm) but also provides clear images of neuronal spines, making significant contributions to the field of neuroscience.

To address this challenge, this thesis focuses on developing a novel two-photon fluorescence miniaturized microscope system capable of imaging cellular activity in deep brain regions of mice, nicknamed NTUminiscope. Given that we are utilizing a point-scanning two-photon imaging system and need to reduce the overall weight of the microscope, we selected a miniaturized scanning mirror (MEMS) in combination with a gradient-index lens (GRIN lens) to reach deeper brain regions. The system design and implementartion of NTUminiscope is divided into four main sections: optical design using Zemax software, which simulates a theoretical 2P resolution of 1.14 μm with a field of view of 123 μm²; optomechanical structural design using Shapr3D software and 3D printing technology, and the development of unique optical alignment techniques (the "hole-cage alignment method") suited for this miniaturized system. Additionally, a C++/CLI graphical user interface for high-speed data acquisition and real-time image processing using a graphics card.

For the microscope to visualize neuronal images, it must achieve sub-micron level lateral resolution. Furthermore, to observe neural functional imaging in freely moving mice in the future, the system needs to be based on fiber optics for laser light transmission and fluorescent signal collection. It is also worth mentioning that before conducting in vivo experiments, viruses (AAV9-syn-GCamp6f) need to be introduced into the targeted brain regions, enhancing the collection and observation of fluorescent images.

The first-generation NTU miniScope is divided into two main modules: the "imaging module" and the "base module, which is to be implanted into the mouse brain." These two modules are magnetically attached for easy positioning and detachment, allowing the system to partially disconnected from the animal as needed. Based on our optical design, the base module can be swapped depending on the specific brain region we aim to observe on any given day. In summary, this innovative NTU miniScope design weighs just 5 grams, with dimensions of 22 mm x 6 mm x 21 mm, an imaging frame rate of 20 frames per second, and a 2P lateral resolution of 1.292 ± 0.012 microns. The specialized optomechanical design enables functional neural imaging of the substantia nigra pars compacta.