SummaryHere, we present a protocol to observe the three-dimensional surface of nuclear pore complexes (NPCs) of vertebrate early embryos by field emission scanning electron microscopy (FESEM). We describe steps from zebrafish early embryo collection and nuclei exposure to FESEM sample preparation and final NPC state analysis. This approach provides an easy way to observe surface morphology of NPCs from the cytoplasmic side. Alternatively, additional purification steps after nuclei exposure supply intact nuclei for further mass spectrometry analysis or other utilization.For complete details on the use and execution of this protocol, please refer to Shen et al.1