体内单神经元形态与受限引发转化
Single neuron morphology in vivo with confined primed conversion.
作者信息
Mohr M A, Pantazis P
机构信息
Eidgenössische Technische Hochschule Zurich (ETH Zurich), Basel, Switzerland.
出版信息
Methods Cell Biol. 2016;133:125-38. doi: 10.1016/bs.mcb.2015.12.005. Epub 2016 Feb 28.
Unraveling the structural organization of neurons can provide fundamental insights into brain function. However, visualizing neurite morphology in vivo remains difficult due to the high density and complexity of neural packing in the nervous system. Detailed analysis of neural morphology requires distinction of closely neighboring, highly intricate cellular structures such as neurites with high contrast. Green-to-red photoconvertible fluorescent proteins have become powerful tools to optically highlight molecular and cellular structures for developmental and cell biological studies. Yet, selective labeling of single cells of interest in vivo has been precluded due to inefficient photoconversion when using high intensity, pulsed, near-infrared laser sources that are commonly applied for achieving axially confined two-photon (2P) fluorescence excitation. Here we describe a novel optical mechanism, "confined primed conversion," which employs continuous dual-wave illumination to achieve confined green-to-red photoconversion of single cells in live zebrafish embryos. Confined primed conversion exhibits wide applicability and this chapter specifically elaborates on employing this imaging modality to analyze neural morphology of optically targeted single neurons in the developing zebrafish brain.
揭示神经元的结构组织有助于深入了解大脑功能。然而,由于神经系统中神经组织的高密度和复杂性,在体内观察神经突形态仍然具有挑战性。对神经形态的详细分析需要区分紧密相邻、高度复杂的细胞结构,如具有高对比度的神经突。绿到红的光转换荧光蛋白已成为发育和细胞生物学研究中光学突出分子和细胞结构的有力工具。然而,由于在使用通常用于实现轴向受限双光子(2P)荧光激发的高强度、脉冲、近红外激光源时光转换效率低下,体内感兴趣的单个细胞的选择性标记一直受到限制。在这里,我们描述了一种新的光学机制,即“受限预转换”,它采用连续双波照明来实现活斑马鱼胚胎中单个细胞的受限绿到红的光转换。受限预转换具有广泛的适用性,本章具体阐述了如何利用这种成像方式来分析发育中的斑马鱼大脑中光学靶向的单个神经元的神经形态。