Abstract Combining live imaging with single-cell sequencing is a powerful approach for gaining comprehensive understanding of individual cell behaviors within complex biological systems. However, existing approaches are limited in throughput, rely on computational data alignments that are prone to bias, or require analysis of isolated cells. The goal of this Fast-Track SBIR application is to develop a novel cellular barcoding technology that will enable researchers to conduct both standard live-cell imaging and high- throughput single-cell sequencing, and then align the two sets of data unambiguously according to the cell barcodes. The proposed approach uses dual-barcoded laser particles (LPs) with unique pairs of optical emission spectra and oligonucleotide sequences. These barcodes can be identified either optically during imaging or by single-cell sequencing. Built on promising preliminary data, Phase I of this project aims to demonstrate the feasibility of dual-barcoded LPs on a small scale. Upon success of Phase I, Phase II will streamline and scale up the fabrication of the LPs and test and validate the technology towards commercialization. The optical barcode readout requires a high-resolution spectrometer and a pump laser. In Phase II, add-on hardware modules and cell-tracking software will be developed and tested to be compatible with existing commercial microscopes, making it easier for researchers to adopt the technology. Additionally, Phase II research aims to demonstrate the usefulness of imaging-sequencing data via a pilot study of cancer cells. Furthermore, potential risks and solutions for the technology will be explored, including the long-term effects of LPs on cell gene expression patterns. Overall, the outcome of this Fast- Track project will be significant technological advancements to the field of single-cell analysis for both basic research and drug discovery.