RainBar: Optical Barcoding for Pooled Live-Cell Imaging with Single-Cell Resolution.

ABSTRACT: High-throughput pooled screening has advanced functional genomics, but most existing methods rely on endpoint sequencing and are blind to dynamic, time-resolved phenotypes. We developed RainBar (Rainbow Barcodes), an optical barcoding system that supports pooled live-cell imaging with single-cell resolution. RainBar uses lentiviral co-delivery of spectrally distinct nuclear and cytoplasmic fluorescent proteins to encode up to 64 unique perturbations per well. To mitigate barcode recombination and improve decoding accuracy, we employed single-template viral production, codon diversification, and a ratio-based spectral unmixing pipeline tailored to overlapping fluorophores. An inverted cytoplasmic segmentation approach and multilayer perceptron classifier enabled accurate barcode identificat

SUBMITTER: Mosadeghi R 

PROVIDER: S-EPMC12637547 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature