A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.
Chromatin/chemistry , Microscopy, Fluorescence/methods , Mouse Embryonic Stem Cells/metabolism , Single Molecule Imaging/methods , Animals , Cell Line , Chromatin/metabolism , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mice , Mouse Embryonic Stem Cells/ultrastructure , Photobleaching
Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000-100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.
Chromatin/ultrastructure , Chromosomes/ultrastructure , Molecular Biology/methods , Molecular Conformation , Mouse Embryonic Stem Cells , Optical Imaging/methods , Animals , Cells, Cultured , Imaging, Three-Dimensional/methods , Mice , Single-Cell Analysis/methods
The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.
Chromatin Assembly and Disassembly , Genome , Molecular Imaging/methods , Nucleosomes/chemistry , Single-Cell Analysis/methods , Animals , CCCTC-Binding Factor , Cell Cycle Proteins/metabolism , Chromatin Assembly and Disassembly/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Mammalian/chemistry , Chromosomes, Mammalian/genetics , Chromosomes, Mammalian/metabolism , DNA/chemistry , DNA/genetics , DNA/metabolism , Enhancer Elements, Genetic , G1 Phase , Gene Expression Regulation , Gene Regulatory Networks , Genome/genetics , Haploidy , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Mice , Models, Molecular , Molecular Conformation , Molecular Imaging/standards , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Promoter Regions, Genetic , Repressor Proteins/metabolism , Reproducibility of Results , Single-Cell Analysis/standards , Cohesins
A multi-layer device, combining hydrodynamic trapping with microfluidic valving techniques, has been developed for on-chip manipulation and imaging of single cells and particles. Such a device contains a flow layer with trapping channels to capture single particles or cells and a control layer with valve channels to selectively control the trap and release processes. Particles and cells have been successfully trapped and released using the proposed device. The device enables the trapping of single particles with a trapping efficiency of greater than 95%, and allows for single particles and cells to be trapped, released and manipulated by simply controlling corresponding valves. Moreover, the trap and release processes are found to be compatible with biological samples like cells. Our device allows stable immobilisation of large numbers of single cells in a few minutes, significantly easing the experiment setup for single-cell characterisation and offering a stable platform for both single-molecule and super-resolution imaging. Proof-of-concept super- resolution imaging experiments with mouse embryonic stem cells (mESCs) have been conducted by exploiting super-resolution photoactivated localisation microscopy (PALM). Cells and nuclei were stably trapped and imaged. Centromeres of â¼200 nm size could be identified with a localisation precision of <15 nm.
