Confocal Live Imaging of Reproductive Organs Development in <em>Arabidopsis</em>

Understanding how multicellular organisms are shaped requires high-resolution, quantitative data to unravel how biological structures grow and develop over time. In recent years, confocal live imaging has become an essential tool providing insights into developmental dynamics at cellular resolution in plant organs such as leaves or meristems. In the context of flowers, growth tracking has primarily been limited to sepals, the outermost floral organs, or the post-fertilization gynoecium, which are easily accessible for microscopy. Here, we describe a detailed pipeline for the preparation, dissection, and confocal imaging of the development of internal reproductive floral organs of <i>Arabidopsis thaliana</i> including both the stamen and gynoecium. We also discuss how to acquire high-quality images suitable for efficient 2D and 3D segmentation that allow the quantification of cellular dynamics underlying their development. Key features • Fine dissection of tiny and tightly enclosed floral organs. • Confocal live imaging method allowing long-term observation of plant reproductive morphogenesis. • Assessing the quality of acquired images for efficient segmentation at cellular resolution in 2D and 3D.