Label-Free Super-Resolution Imaging of Chromatin Structure and Dynamics

Higher-order chromatin structure is crucial to gene regulation. Super-resolution optical microscopy is among the key technologies that are used to elucidate the organization of chromatin. The importance of nanoscopic imaging is underscored by the 2014 Nobel Prize in Chemistry. However, the potential of this technology can be immensely enhanced. Existing methods rely on fluorescent labeling of biomolecules. Because it is virtually impossible to label every nucleotide, the resolution of the most widely used nanoscopy technologies is limited to a few tens of nanometers, domains that may contain tens of thousands base pairs. Bringing the resolution down to the size of a single nucleosome, approximately 1 nm, is the next frontier.

This talk will discuss a new label-free optical nanoscopy capable of molecular imaging with 6 nm resolution. The technique is based on a newly discovered physical effect: most biopolymers such as DNA and proteins, which until recently had been considered “dark” in the visible regime, exhibit stochastic photoswitchable autofluorescence when illuminated by visible light due to the ground-state depletion (GSD) recovery phenomenon. The technology combines time-resolved stochastic photon localization with simultaneous spectroscopic molecular-signature-carrying intrinsic fluorescence detection, referred to as spectroscopic intrinsic-contrast photon-localization optical nanoscopy (SICLON). SICLON is able to image DNA, chromatin, and proteins without labeling. Importantly, most significant biomolecules (DNA, proteins) have distinct spectral signatures, which carry information about nucleotide and amino acid composition; thus SICLON enables molecular recognition.

Genomes are highly complex hierarchical assemblages. At the most basic (~1 nm) level, the linear genetic code has been mostly decoded. At the next (~10 nm) level is the histone code, which regulates the expression of the individual histone-associated genes. At an even higher level, supra-nucleosomal chromatin structure plays a critical role in the regulation of the global pattern of gene expression, impacting upon thousands of genes. The dysregulation of chromatin compartmentalization has been implicated in many diseases including cancer, atherosclerosis, and neurodegenerative diseases. Decoding chromatin compartmentalization is the next frontier. The talk will discuss the insights gained from label-free optical nanoscopy into the chromatin topology as a key code regulating global patterns of gene expression.