Project Summary/Abstract Cells are the basic unit of life. Cells are very dynamic: they change over time and locations, respond to different environments, and interact with other cells. Over the past decade, single-cell biology has witnessed enormous growth owing to massive technical advances, such as single-cell sequencing, multi-omics, and spatial omics. However, obtaining dynamic dimensions of live cells along with their multi-omic information at the single-cell resolution is currently difficult and certainly not possible on large scales. Here, we propose a novel cell barcoding technology that has the potential to enable us to collect live information of cells and connect the data to their detailed omics information. This technology makes use of laser particles (LPs) with unique optical barcodes for >100,000 channels, each containing a unique DNA barcode. The “dual-barcoding” will allow us to optically track live cells under a microscope while they are in their natural environment or in culture, acquire their live information, harvest the cells, acquire the omics information of the same cells by droplet-based next-generation single-cell sequencing, and then combine the live imaging and omics data at the single cell resolution. Furthermore, our technique can be upgraded to multi-omics modalities, combining multiple layers of information from the genome, epigenome, transcriptome, and proteome, together with morphological, locational, functional, and behavioral data. We will apply the method to study sentinel lymph node (SLN) metastasis of cancer cells in vivo. The acquired in vivo single-cell imaging and multi-omics data will provide an unprecedented picture of the cancer cell lymphatic metastasis process. This project has two specific aims. Aim 1 will develop an optical-and- DNA “dual” barcoding strategy for droplet-based single-cell sequencing. Aim 2 will apply the method to study breast cancer SLN metastasis in vivo. During the K99 period, the applicant will receive additional training to expand her experience and shape her independence in the following areas: (1) LPs and optical barcoding, (2) LP imaging and in vivo mouse imaging, and (3) single-cell sequencing and multi-omics. This proposal is under the combined mentorship of Dr. Andy Yun (LP technology, optics, and imaging) and Dr. Ralph Weissleder (cancer biology, in vivo imaging, and system biology), and a team of experts as advisors for single-cell sequencing and bioinformatics. The interdisciplinary research environment at Massachusetts General Hospital and Harvard Medical School will significantly facilitate the proposed study. If successful, the proposed study will offer a new paradigm for “dynamic” single-cell analysis, with unprecedented speed and throughput, enabling multi-omics modalities for the profiling of proteins, RNAs, and DNAs at the single-cell level, together with cells’ dynamic phenotype information, enable spatial-omics profiling at the 3D resolution without the need f...