由於腸道組織神經結構呈現細長纖維狀，標準的二維平面切片機觀察技術並不能提供一個廣泛的三維神經影像網絡。在此項研究中，我們整合了免疫染色法、組織透明技術，以及共軛焦顯微鏡技術去發展一個不需要切片機即可看到老鼠腸道組織三維神經網絡的方式。利用福馬林固定與Triton X-100作用後的組織樣本，運用廣泛的PGP9.5神經標記與交感神經有關的Tyrosine hydroxylase，以及神經膠質標記S100□作為染色的標誌。並藉由將組織浸泡在FocusClearTM中能增進光的穿透度，能更清楚觀察在厚片組織中免疫染色法所標記到的神經網絡，增進光的穿透度能使得我們得到高解析度的神經網絡影像。除了組織透明技術之外，輔以Triton X-100的使用，使得組織樣本的通透度更好，能增加免疫染色影像的訊號對雜訊的比例，提升神經網絡影像的品質。我們提供了一個便捷且直接的方式來取得組織中連續性的神經影像訊息，這項研究能改善舊有的二維平面腸道神經的觀察模式，以獲得一個三維空間的神經網絡，因而更能幫助了解腸道組織的生理訊號以及幫助疾病的判斷。

Due to the fibrous structure of the enteric nervous system, standard microtome-based planar microscopy cannot provide an integral view of the 3-dimensional (3-D) neural networks. In this research, we integrated immunostaining, optical clearing, and confocal microscopy to develop a microtome-free method for in-depth, 3-dimensional (3-D) visualization of the mouse intestinal innervation. The pan-neuronal marker PGP9.5, the sympathetic neuronal marker tyrosine hydroxylase, and the glial marker S100□ were used as the staining targets after paraformaldehyde fixation and Triton X-100 permeabilization of the mouse ileum. The immunolabeled nerve tissues were immersed in the optical-clearing solution, FocusClearTM, to improve light transmission to facilitate deep-tissue confocal microscopy. The improved photon penetration resulting from optical clearing of the tissues led to high-resolution image acquisition of the neural network. In addition to optical clearing, tissue permeabilization by Triton X-100 was essential to increase the signal-to-noise ratio in immunostaining/imaging. We provide a convenient and straightforward method to acquire a continuous flow of the anatomic neural information in situ. This approach will change our conventional planar view of the enteric nervous system into a 3-D panorama to better understand the intestinal physiology and diseases.

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