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Live imaging is the gold standard for determining how cells give rise to organs. However, tracking many cells across whole organs over large developmental time windows is extremely challenging. In this work, we provide a comparably simple method for confocal live imaging entireArabidopsis thalianafirst leaves across early development. Our imaging method works for both wild-type leaves and the complex curved leaves of thejaw-1Dmutant.

We find that dissecting the cotyledons, affixing a coverslip above the samples and mounting samples with perfluorodecalin yields optimal imaging series for robust cellular and organ level analysis. We provide details of our complementary image processing steps in MorphoGraphX software for segmenting, tracking lineages, and measuring a suite of cellular properties. We also provide MorphoGraphX image processing scripts we developed to automate analysis of segmented images and data presentation.

Our imaging techniques and processing steps combine into a robust imaging pipeline. With this pipeline we are able to examine important nuances in the cellular growth and differentiation ofjaw-Dversus WT leaves that have not been demonstrated before. Our pipeline is approachable and easy to use for leaf development live imaging.