Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is an electron microscope (EM) sample preparation technique which allows for high-resolution visualization of membrane proteins with high sensitivity. However, image acquisition of specific replica profiles such as synapses in a large field of EM view needs a valid experience and a long time for manual searching. Here, we describe how to utilize deep learning for automatizing image acquisition of specific profiles of interest in replica samples. This protocol facilitates the labor-intensive collection of EM images, in particular for rare profiles. We provide instructions for using SerialEM image acquisition software in conjunction with object detection by our newly developed deep learning software DarEM, to automatically acquire tilt series of all synapses in a selected region. We then show how to perform a mostly automated analysis of gold particle labeling in the acquired images by utilizing Darea software. 123-137 123-137 Springer Nature