用於透明化完整鼠腦之超高解析度成像之8mm工作距離雙光子顯微鏡

Abstract

神經網絡結構解析一直是腦科學中十分重要的議題，當中大腦神經元間之連結體在神經元之間之連結扮演著十分重要之角色。而大腦之神經結構與腦部功能會互相影響，但神經結構以及腦部功能之詳細關係依然需要更多的研究進行驗證，然而，大腦中的細胞密度以及細胞多樣性使神經網絡的解釋變得極其困難。而螢光蛋白基因轉殖小鼠模型可以利用特殊的螢光標記來剋服上述困難，因此螢光蛋白基因轉殖小鼠模型的出現使得光學顯微鏡在神經網絡結構解析的相關研究中扮演著極為重要的角色。目前，透過結合透明化技術以及層光顯微技術 (LSM)已實現單細胞解析度之鼠腦神經網絡重建。 然而，單細胞解析度並不足以解析鼠腦神經網絡。系統必須到達微米等級或以上的解析度才足以解析鼠腦神經網絡之細微結構。由於雙光子顯微鏡的在深層成像時可以維持相對高的信號背景比 (SBR)以及雙光子所使用的長波長光源能有效減少散射，雙光子顯微鏡近年來已被廣泛應用在厚樣品成像。雖然透明化透過減少生物樣品中的折射率差異來減少樣品對光的吸收和散射，藉此提升光學的穿透深度以及解析度，但生物樣品之光學穿透深度目前國際上還沒有突破6mm。 由於成年老鼠之完整大腦厚度大約6-7mm，要解析完整鼠腦之神經網絡結構必須使鼠腦的光學穿透深度至少大於6mm且同時保有微米等級的解析度。本研究建立了能達8mm工作距離之極高解析度雙光子顯微鏡，並用於透明化、完整鼠腦之神經網絡三維成像。本研究利用了中心波長為1070nm, 脈衝寬度為 ~55fs之飛秒雷射作為激發光源以減少光的散射以及散射效率的差異。為了確保穿透深度能超過成年鼠腦之大小以及優化光學系統，本研究所建立之系統選用了有矯正環、工作距離為8mm之物鏡 (NA = 1, 後光圈直徑為14.4mm)。透過研究物鏡矯正環對影像之影響、不同影像平均時間對訊號雜訊比、訊號背景比和對比度之影響、樣品以及浸泡溶液之折射率匹配，本研究發現此具8mm工作距離之雙光子顯微鏡系統之光學穿透深度可達到至少7.1mm，且在此對應之穿透深度達到1.15±0.17微米之系統解析度(1/e^2雙光子點函數之半徑)。

Revealing the 3D structure of neural networks plays an important role in neuroscience. Researchers believe the neuron structure and the brain function affect each other. However, the relation between the connectome in the brain and the brain function still needs to be studied. Due to the cell density and cell diversity, analyzing the neural network of the mouse brain is a huge challenge. The fluorescent transgenic mouse model provides a solution for this limitation, and fluorescence microscopy has become an irreplaceable tool for investigating the neuron network. Reconstruction of the neuron network with single-cell resolution was achieved by combining tissue clearing and a light-sheet microscope. However, to resolve the neuron network of the brain, micron resolution is required for the fine structure of the connectomes. Two-photon microscope has been widely used in deep tissue imaging as longer wavelength reduces the scattering of the light, and the physical mechanism of two-photon excitation maintains high signal-to-background ratio of the images for deep brain imaging. Although tissue clearing reduces the absorption and scattering of the biological samples by minimizing the difference of refractive index in the samples, the penetration depth is usually limited to ~6mm for whole brain imaging. To visualize the neuron network of the intact mouse brain, the minimum penetration depth should be larger than 6mm and preserve microns resolution in such penetration depth. In this study, an ultra-high resolution two-photon microscope with 8mm working distance was developed for visualizing the 3D structure of a cleared, intact mouse brain. A femtosecond laser with central wavelength 1070nm and ~55fs pulse width was used as the excitation source to reduce the scattering of light and minimize the difference in scattering efficiency. An 8mm working distance objective lens with correction collar (NA = 1 and rear aperture =14.4mm) was used to extend the working distance and optimize the optics in two-photon scanning microscope. The effects of the correction collar and the SNR, SBR, and contrast ratio with different numbers of frame averaging were evaluated to study image quality of the system after the light passes through a cleared, intact mouse brain. The image quality and the penetration depth of different immersion media for imaging and cleared mouse brain were studied. We discovered that ≥7.1mm penetration depth and 1.15±0.17μm resolution (defined as the 1/e^2 beam radius of the two-photon point spread function) can be achieved in such penetration depth for the ultra-high resolution two-photon microscope.