Project Summary The broad objective of this proposed work is to advance single-cell analysis by introducing novel cell-barcoding technologies. Understanding cells is a cornerstone of life sciences. Single-cell sequencing led to a paradigm shift in our approach to analyze cells from ensembles to individual cells. Unfortunately, the current single-cell analysis techniques are almost exclusively static, performed ex vivo, and thus cannot easily probe the dynamic cellular processes. Beyond static analysis, the next important step is to add more dimensions: everything that makes a cell a living entity, including temporal changes, spatial movement, interactions with other cells in vivo. This project will develop laser particles (LPs), which can be read optically in real time and repeatedly as needed, for multi-dimensional single-cell analysis. With the optical barcode, it is possible to acquire data from a cell at different times, locations and apparatuses, and compile the data to produce a full account of the cell. A combination of LPs with oligonucleotide barcodes enables a breakthrough approach acquiring large-scale data from in vivo physiology to molecular omics using multiple instruments in series. This project is organized in three specific aims. Aim 1 develops large-scale LP barcodes combined with oligo barcodes. Aim 2 constructs several instruments integrating LP barcode readers into commercial platforms. Aim 3 involves validation studies to demonstrate the immediate utilities of the new technologies. The optical barcoding is expected to make a paradigm change in single-cell analysis. This will transform the way multi-dimensional single-cell analysis is used for scientific discovery and diagnostic and therapeutic applications in healthcare.