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Abstract A major challenge in cell and developmental biology is the automated identification and quantitation of cells in complex multilayered tissues. We developed CytoCensus: an easily deployed implementation of supervised machine learning that extends convenient 2D “point- and-click” user training to 3D detection of cells in challenging datasets with ill-defined cell boundaries. In tests on these datasets, CytoCensus outperforms other freely available image analysis software in accuracy and speed of cell detection. We used CytoCensus to count stem cells and their progeny, and to quantify individual cell divisions from time-lapse movies of explanted Drosophila larval brains, comparing wild-type and mutant phenotypes. We further illustrate the general utility and future potential of CytoCensus by analysing the 3D organisation of multiple cell classes in Zebrafish retinal organoids and cell distributions in mouse embryos. CytoCensus opens the possibility of straightforward and robust automated analysis of developmental phenotypes in complex tissues. Summary Hailstone et al . develop CytoCensus, a “point-and-click” supervised machine-learning image analysis software to quantitatively identify defined cell classes and divisions from large multidimensional data sets of complex tissues. They demonstrate its utility in analysing challenging developmental phenotypes in living explanted Drosophila larval brains, mammalian embryos and zebrafish organoids. They further show, in comparative tests, a significant improvement in performance over existing easy-to-use image analysis software. <jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="" xlink:href="137406v4_ufig1" position="float" orientation="portrait" /> Highlights <jats:list list-type="bullet"><jats:list-item> CytoCensus: machine learning quantitation of cell types in complex 3D tissues <jats:list-item> Single cell analysis of division rates from movies of living Drosophila brains in 3D <jats:list-item> Diverse applications in the analysis of developing vertebrate tissues and organoids <jats:list-item> Outperforms other image analysis software on challenging, low SNR datasets tested

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