Randomized Spatial Barcoding for Time-Lapse Flow Cytometry

Tracking suspended cells over multiple time points at the single-cell level remains challenging because existing flow-based methods cannot preserve cell identity while maintaining high throughput. Here, we present RASPBerry, a hydrogel-based spatial barcoding platform for time-lapse flow cytometry. RASPBerry generates unique barcodes by randomly co-encapsulating fluorescent beads with individual cells in hydrogel droplets, eliminating the need for predefined barcode patterns or specialized optical instrumentation. We integrate RASPBerry with acoustofluidic imaging flow cytometry to enable time-lapse imaging flow cytometry of suspended cells. The platform identifies more than 17,000 hydrogel droplets with 99.8% matching accuracy. We further demonstrate time-lapse tracking of more than 10,000 suspended cells and quantify stress-induced nuclear morphological changes in more than 5,000 individual cells. RASPBerry provides a simple, scalable, and broadly accessible strategy for time-lapse imaging flow cytometry, expanding the capability for dynamic single-cell analysis of suspended cells.