AbstractThree dimensional segmentation and analysis of dendritic spines involve two major challenges: 1) how to segment individual spines from the dendrites and 2) how to quantitatively assess the morphology of individual spines. We developed a software named 3dSpAn to address these two issues by implementing our previously published 3D multiscale opening algorithm in shared intensity space and using effective morphological features for individual dendritic spine plasticity analysis. 3dSpAn consists of four modules: Preprocessing and ROI selection, Intensity thresholding and seed selection, Multiscale segmentation and Quantitative morphological feature extraction. We show the results of segmentation and morphological analysis for different observation methods, including in vitro and ex vivo imaging with confocal microscopy, and in vivo samples, using high-resolution two-photon microscopy. The software is freely available, the source code, windows installer, the software manual and video tutorial can be obtained from: https://sites.google.com/view/3dSpAn/.