Basic Image Analysis and Manipulation in ImageJ/Fiji

Abstract Microscopy‐generated images contain quantitative information that can be extracted using a variety of analysis methods and automated pipelines. In this protocol, basic aspects of image analysis are outlined, including software installation, data import, image processing functions, and analytical tools that can be used to extract information from microscopy data using the open‐source software ImageJ/Fiji. Step‐by‐step protocols for analyzing objects (i.e., nuclei) in a fluorescence image are included, focusing on segmentation routines. © 2023 Wiley Periodicals LLC.