We propose to visualizing chromosome structure and dynamics in single cells by a combination of two approaches: sequential FISH and CRISPR imaging. seqFISH methods developed in the Cai lab allows multiple nucleic acid species to be detected in single cells using sequential rounds of hybridization to barcode transcripts and genomic loci. The Cai lab will extend this method to DNA FISH to tag 100 different loci in single cells, providing a snapshot of the structure of the chromosome in individual cells. To image the dynamic of these loci, the Qi lab has developed CRISPR imaging tools which uses a catalytically dead Cas9 labeled with GFP that can be targeted to particular chromosomal loci by gRNAs. We will discuss the practical implementations and solutions to potential pitfalls. We will investigate how much heterogeneity exist in chromosome structures in single cells and how they are correlated to functional measures of transcriptional and epigenetic states of individual cells. Mouse embryonic stem cells are a natural system to examine heterogeneity in chromosome structure, because mESCs have been extensively studied for transcriptional level heterogeneity. Thus, it offers the best platform to examine the relationship between chromosomal organization and transcriptional regulation. In addition, seqFISH imaging allows us to validate the Hi-C results independently. We can examine whether topological associated domains and chromosomal territories are observed in FISH as they have in HiC experiments. We will characterize the distribution of the heterogeneities in the chromosome structures at various length scales in single cells and generate unprecedented direct images of the chromosomes. The methods developed in this project can be easily disseminated for general use. FISH has been used extensively for chromosome studies. Developed protocols for seqFISH can be readily adopted by any lab with experience with in situ hybridization and microscopy. All protocols developed in the project will be made available publicly.