Single-Molecule Fluorescence Microscopy Methods in Chromatin Biology

Abstract

Classical biochemical and molecular genetic techniques have provided fundamental insights into genomic functions and chromatin organization. Recent advances in the development of fluorescence microscopy imaging methods as well as new labeling techniques enable visualizing chromatin structure and tracking dynamics of transcription factors within mammalian cells at a single-molecule level. Single particle tracking of transcription factors within cells provides insight into how transcription factors explore the complex nuclear environment and assemble on their target sites. Superresolution microscopy based on single-molecule centroid determination has been applied to map chromatin structure at a nucleosomal resolution. This chapter outlines recent advances in the application of single molecule-based fluorescence microscopy techniques in chromatin biology within mammalian cells.

Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells

“…Direct observation of the molecular processes in living cells is critical for quantitatively understanding of how biological systems function. Recent advances in fluorescence microscopy and fluorescent labelling enable to visualize trajectories of individually single molecules in living cells, providing insights about dynamic interactions and assemblies of biological molecules ( Kusumi et al ., 2014 ; Liu et al ., 2015 ; Tatavosian et al ., 2015 ; Cuvier and Fierz, 2017 ). Specific labelling of biomolecules with fluorophores is the key for fluorescence single-molecule imaging.…”