Determining the Age of Every Cell Within Each Budding Yeast Microcolony Combining Single-Cell Microencapsulation With Confocal Microscopy.

Isogenic populations of Saccharomyces cerevisiae exhibit significant proliferative heterogeneity, with individual cells within a clonal culture displaying divergent growth rates and metabolic states. Investigating the origins of this variation requires a method to reconstruct the individual histories of cells within the population. This protocol describes a method for single-cell microencapsulation in alginate microspheres to create a physically stable, traceable, three-dimensional genealogical environment. By utilizing the alginate matrix to prevent daughter cell migration, the replicative history of a founder cell can be mathematically reconstructed. This is achieved by correlating the total cell count (N) within a developed microcolony with the total number of accumulated bud scars (n) visualized via confocal microscopy. Key features • Enables non-destructive 3D reconstruction of yeast genealogies, preserving spatial architecture and mapping all division events from a single founder cell. • Implements a robust mathematical formula to determine the founder's replicative age, linking past mitotic history to current microcolony growth dynamics. • Combines Flow Focusing^® microencapsulation with precise confocal Z-stack analysis to review lineage-specific phenotypic heterogeneity in isogenic populations. • Ideal for studying transgenerational inheritance and growth-rate diversification, providing a traceable, three-dimensional genealogical ecosystem for single-cell research.