Knott Graham W
Cold Spring Harb Protoc. 2013 Jun 1;2013(6):542-50. doi: 10.1101/pdb.prot075127.
The ability to observe axons and dendrites with transmission electron microscopy (EM) after they have been previously imaged live with laser-scanning microscopy is a useful technique to study their synaptic connectivity. This protocol provides a detailed method by which neurons that were imaged in a live brain or slice culture can be reimaged using EM. First, brain tissue expressing green fluorescent protein (GFP) is chemically fixed. Then, an immunocytochemistry process is used to render the fluorescent protein electron dense so that it can first be located using light microscopy and then serial thin-sectioned for EM so that the ultrastructure of specific parts of neurites can be analyzed in three dimensions. Patterns of blood vessels observed in the live brain are used to locate the previously imaged neurons. The method described here allows for a complete three-dimensional (3D) reconstruction to be made of the imaged structures from serial electron micrographs.
在用激光扫描显微镜对轴突和树突进行活体成像后，利用透射电子显微镜（EM）观察它们的能力是研究其突触连接性的一项有用技术。本方案提供了一种详细方法，通过该方法可以对在活体脑或脑片培养物中成像的神经元进行EM再成像。首先，对表达绿色荧光蛋白（GFP）的脑组织进行化学固定。然后，采用免疫细胞化学方法使荧光蛋白具有电子密度，以便首先通过光学显微镜定位，然后进行连续超薄切片用于EM，从而可以在三维空间中分析神经突特定部分的超微结构。利用在活体脑中观察到的血管模式来定位先前成像的神经元。这里描述的方法允许从连续电子显微照片对成像结构进行完整的三维（3D）重建。
