Summary Cells carrying the same DNA sequence can exhibit heterogeneous gene expression, leading to phenotypic and functional diversity in both healthy and diseased states. This heterogeneity fundamentally arises at the nucleus level often from different spatio-temporal patterns of genomic organization, which could differentially influence the frequency and strength of the physical interactions among genetic elements related to gene expression. Disruptions in these interaction patterns are often associated with disease, and accordingly, there is a growing interest in advancing tools for identifying abnormal spatial patterns and contact profiles of the genome. DNA fluorescence in situ hybridization (FISH) is intrinsically a single-cell assay and suitable for probing cell-to-cell variation as well as targeted detection of chromosomal interactions. However, the use of DNA FISH for high- throughput and high-resolution proximity detection is presently limited due to the lack of strategies enabling multiplexing and high-specific labeling with low background signal. This project will devise two separate FISH approaches that address the multiplexing and labeling challenges of DNA FISH by making novel use of our lab's recently developed Signal Amplification By Exchange Reaction (SABER) method (Nature Methods, 2019), which can simultaneously increase imaging throughput and multiplexing levels. Specifically, the first aim will introduce a variant of the SABER method that only allows signal amplification upon physical contact between a pair of FISH probes. This method will be optimized for DNA FISH and its wide versatility will be demonstrated in the second aim, in the two separate applications: 1) for high-specific and low-background labeling of short DNA targets and 2) a correction-free (i.e. no channel alignment) one-step colocalization assay for detection of distal DNA sequences in close 3D proximity. The outstanding research environment of the Wyss Institute for Biologically Inspired Engineering at Harvard University will offer numerous resources critical for the successful completion of the proposed goals and ensure the maximum impact of the research given the institute's core focus is on novel technology development and translation. The sponsor of the project, Dr. Peng Yin, and his team who have expertise in developing DNA-based molecular devises, imaging, and experience with chromosomal studies will provide detailed technical support and personalized mentorship. The successful completion of the aims will thus bring new methodologies to the growing scientific community at the interface between chromosome conformation capture (3C) and FISH for cross-validation of contact profiles and will further expand the utility of FISH in potential diagnostic applications.