Abstract
Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions.
Highlights
In spite of a great deal of knowledge already available, the precise higher order spatial organisation of chromatin at the nanoscale is still a subject of an ongoing debate [1,2,3]
In case of YOYO-1 being released from the DNA, we expected that it would stay inside the cell nucleus for tens of minutes because of the constrained diffusion (Supplementary Figure S1)
The DNA structure fluctuation-assisted binding-activated localization microscopy (BALM) approach described in this report made it possible to create single molecule localization images of the distribution of YOYO-1 interacting with nuclear DNA in mammalian cells with a structural resolution in the tens of nanometre range
Summary
In spite of a great deal of knowledge already available, the precise higher order spatial organisation of chromatin at the nanoscale is still a subject of an ongoing debate [1,2,3]. The most plausible models are based on optical microscopy data and suggest the existence of small chromatin domains [4], or on chromosome conformation capture studies suggesting the existence of regions denoted as ‘topologically associated chromatin domains’ (TADs) [5,6] These small chromatin domains ranging in size from hundreds of thousands to millions of base pairs are believed to constitute a basic higher order chromatin organisation unit above the level of the individual nucleosomes, as for instance in DNA replication [7]. Among various techniques that contributed significantly to our current understanding of the genome organisation (reviewed in [3,8,9,10]), the recently emerged methods of super-resolution microscopy deserve a special attention These methods have already been successfully applied to studies of the cell nucleus in situ. Such structural details may provide a solid background for the interpretation of other chro-
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