CTransformer: Deep-transformer-based 3D cell membrane tracking with subcellular-resolved molecular quantification

Abstract Deep learning segmentation and fluorescence imaging techniques allow the cellular morphology of living embryos to be constructed spatiotemporally. These development processes involve numerous molecules distributed at the subcellular scale, such as cell adhesion (E-cadherin), which accumulate at cell-cell interfaces to regulate intercellular connections. However, quantifying molecular distributions within specific subcellular regions across the entire embryo, where cell movement and molecular redistribution occur rapidly, is challenging due to the need for simultaneous cell morphology reconstruction and lineage tracing due to photobleaching and phototoxicity. We report a transformer-based pipeline, CTransformer, that establishes a 4D cellular morphology map before the 550-cell (late) stage. CTransformer constructed 4D cellular morphology atlases, reaching 80% accuracy at the 550-cell stage. Through this advanced architecture, we use only one channel to reconstruct cell morphology and achieve cell tracing. With each cell’s morphology as a reference, the distribution of specific molecules throughout the cell body and at cell interfaces can be quantitatively measured in another fluorescence channel. We apply this methodology to track E-cadherin during embryonic development of the worm Caenorhabditis elegans, from fertilization to gastrulation. Our results reveal that E-cadherin is tightly regulated across individual embryos, both within single cells and at cell-cell interfaces, displaying an anterior-posterior gradient and cell- and lineage-specific patterns. Furthermore, its spatiotemporal heterogeneity influences cell mechanics and embryonic morphogenesis, helping explain how C. elegans achieves stereotypical developmental patterns at cellular resolution. In summary, CTransformer offers a high-throughput approach for measuring molecular distributions at subcellular resolution, providing valuable knowledge for cellular and developmental biology, biophysics, and bioinformatics.