Abstract
Cell barcodes are essential for a wide array of experimental applications, including lineage tracing, genetic screening, and single-cell analysis. An optimal barcode library would provide high diversity, live-cell compatible identification, and simple readout. In this work, we introduce single chain tandem fluorescent protein (sctFP) barcodes, constructed by linking different fluorescent proteins (FPs) into a single polypeptide chain with varied copy numbers. We found that the fluorescence signal intensity ratio at different wavelengths can reliably differentiate sctFPs generated using cnidarian FPs, but not prokaryotic FPs that require exogenous cofactors. The sctFPs enable the multiplexing of genetically encoded fluorescent biosensors, enhancing current biosensor multiplexing methods through a simplified imaging and analysis pipeline that support high-throughput applications. Their robust spectral profiles are compatible with a broad range of biosensor types. Using sctFPs, we demonstrate simultaneous tracking of various signaling activities with biosensors of different spectral properties. Together, this strategy provides a robust and scalable method for barcoding cells across diverse experimental contexts.