Combining FISH with localisation microscopy: Super-resolution imaging of nuclear genome nanostructures

Abstract

The optical resolution of conventional far field fluorescence light microscopy is restricted to about 200 nm laterally and 600 nm axially and has been thought for many decades to be an insurmountable barrier for the quantitative spatial analysis of cellular and hence also nuclear constituents. Novel approaches in light microscopy have now overcome this barrier. Here, we report on a special method of localisation microscopy, spectral precision distance/ position determination microscopy and its combination with fluorescence in situ hybridization to analyse the spatial distribution of specific DNA sequences in human cell nuclei at the macromolecular optical resolution level. As an example, repetitive DNA sequence DYZ2 located within the heterochromatin region on human chromosome Yq12 was labelled with clone pHY2.1. Between 300 and 700 singleprobe molecules were resolved in individual chromatin domains, corresponding to a detected molecule density around 500/μm 2 , i.e., many times higher than resolvable by conventional fluorescence microscopy. A mean localisation accuracy of about 20 nm indicated a mean optical resolution in the 50 nm range. Beyond new perspectives for light microscopic studies of specific chromatin nanostructures, this may open a new avenue towards the general analysis of copy number of specific DNA sequences in small regions of individual interphase nuclei.

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

“…However, commercially-available FISH-probes are delivered in a ready-to-use buffer mix that is adapted to protocols including heat denaturation [5]. Moreover, the longer such a BAC-probe is, the more it requires a denaturation of a longer part of the DNA strand, which only seems to possible by heat or harsh chemical treatment [58]. …”

Super-resolution fluorescence imaging of chromosomal DNA

“…It also seems likely that this strategy preferentially labels chromatin regions that have high replication-independent histone turnover (i.e., transcriptionally active regions; see Supplementary note 1). Fluorescence in situ hybridization (FISH) has also been combined with SMLM to visualize specific regions of DNA containing repeated sequences (Weiland et al, 2011); although such approaches appear extremely promising, they are still in their early phases of development. Similar labeling strategies have been used in combination with structured illumination microscopy (SIM) in fixed cells (Schermelleh et al, 2008) and stimulated-emission depletion microscopy (STED) on isolated DNA (Persson et al, 2011), and have yielded resolutions of 100 and 40 nm, respectively.…”

“…Analysis of histone distributions have been used to provide evidence for looping-based models of chromatin organization (Bohn et al, 2010) and for the exclusion of transcription sites from the heterochromatic centers of chromosome territories (Markaki et al, 2010). FISH-based techniques have revealed structural features of heterochromatic and centromeric regions of chromosomes Weiland et al, 2011). SIM-based strategies have visualized surprising substructure in mitotic chromosomes (Schermelleh et al, 2008).…”

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

“…Therefore, SRM has opened perspectives for high-resolution optical genome mapping [34]. For example, DNA repeats in the Yq12 heterochromatin region of the human genome have been studied [35]. The number of trinucleotide repeats in the 5’-untranslated region of the FMR1 gene has been counted [36].…”