Bosch Miquel, Castro Jorge, Sur Mriganka, Hayashi Yasunori
RIKEN-MIT Neuroscience Research Center, Saitama, Japan.
The Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA.
Methods Mol Biol. 2025;2910:145-175. doi: 10.1007/978-1-0716-4446-1_10.
Synapses learn and remember by persistent modifications of their internal structures and composition but, due to their small size, it is difficult to observe these changes at the ultrastructural level in real time. Two-photon fluorescence microscopy (2PM) allows time-course live imaging of individual synapses but lacks ultrastructural resolution. Electron microscopy (EM) allows the ultrastructural imaging of subcellular components but cannot detect fluorescence and lacks temporal resolution. Here we describe a combination of procedures designed to achieve the correlated imaging of the same individual synapse under both 2PM and EM. This technique permits the selective stimulation and live imaging of a single dendritic spine and the subsequent localization of the same spine in EM ultrathin serial sections. Landmarks created through a photomarking method based on the 2-photon-induced precipitation of an electrodense compound are used to unequivocally localize the stimulated synapse. This technique was developed to image, for the first time, the ultrastructure of the postsynaptic density in which long-term potentiation was selectively induced just seconds or minutes before, but it can be applied for the study of any biological process that requires the precise relocalization of micron-wide structures for their correlated imaging with 2PM and EM.
突触通过其内部结构和组成的持续改变来学习和记忆，但由于其尺寸微小，很难在超微结构水平实时观察到这些变化。双光子荧光显微镜（2PM）能够对单个突触进行时间进程的实时成像，但缺乏超微结构分辨率。电子显微镜（EM）可以对亚细胞成分进行超微结构成像，但无法检测荧光且缺乏时间分辨率。在此，我们描述了一系列程序的组合，旨在实现同一单个突触在2PM和EM下的相关成像。该技术允许对单个树突棘进行选择性刺激和实时成像，并随后在EM超薄连续切片中定位同一树突棘。通过基于双光子诱导的电子致密化合物沉淀的光标记方法创建的地标，用于明确地定位受刺激的突触。该技术首次被开发用于成像突触后致密部的超微结构，其中在仅几秒或几分钟前选择性地诱导了长时程增强，但它可应用于任何需要对微米级结构进行精确重新定位以与2PM和EM进行相关成像的生物学过程的研究。
