Correlative Ultrastructural Analysis of Functionally Modulated Synapses Using Automatic Tape-Collecting Ultramicrotome - SEM Array Tomography

To understand synaptic plasticity, correlative microscopic approaches using functional imaging by light microscopy combined with electron microscopy (EM) are necessary. Ultrastructure of synapses can be visualized only by EM, but re-finding the same synapses that were manipulated and imaged by light microscope in the EM is a very laborious and time-consuming task when performing the conventional serial ultrathin-sectioning and observation method with transmission EM. Recently, new imaging applications to obtain volume data sets from brain tissues were introduced using an automatic tapecollecting microtome (ATUM) [1] or a focused ion beam We used an ATUMtome (RMC/Boeckeler) and automatic array tomography software, Atlas AT5 (Zeiss) and established a workflow to investigate ultrastructural changes of synapses. We first performed glutamate uncaging by 2-photon laser microscope to induce/modify spines, and obtained the whole cell image with a confocal microscope. Then the tissues are prepared with conventional pre-embedding immuno-EM using nanogold labeling for EM visualization as explained below.