Combining FISH with localisation microscopy: Super-resolution imaging of nuclear genome nanostructures.
Cell-Line, Cell-Nucleus, Chromosomes-Human-Y, DNA, Fibroblasts, Genome-Human, Heterochromatin, Humans, In-Situ-Hybridization-Fluorescence, Microscopy, Microscopy-Fluorescence, Nanostructures
Chromosome Res 2011 Jan; 19(1):5-23.
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 single-probe molecules were resolved in individual chromatin domains, corresponding to a detected molecule density around 500/mum(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 FISH with localisation microscopy: Super-resolution imaging of nuclear genome nanostructures. Chromosome Res 2011 Jan; 19(1):5-23.