Super-Resolution Microscopy of Chromatin

Abstract

Since the advent of super-resolution microscopy, countless approaches and studies have been published contributing significantly to our understanding of cellular processes. With the aid of chromatin-specific fluorescence labeling techniques, we are gaining increasing insight into gene regulation and chromatin organization. Combined with super-resolution imaging and data analysis, these labeling techniques enable direct assessment not only of chromatin interactions but also of the function of specific chromatin conformational states.

Keywords: DNA; DNA labeling; chromatin; fluorescence; nuclear architecture; single molecule localization microscopy; super-resolution microscopy.

Figure 1

Imaging DNA. (a) Apart from indirect labeling via tagged proteins, DNA can be labeled directly using a number of labeling strategies with DNA specific dyes binding e.g., to the minor or major grove, or in-between the two DNA strands (intercalating dye). (b) Oligonucleotide sequences offer the possibility to attach a removable fluorescently labeled oligoprobe to single stranded DNA, e.g., after a denaturation step [36]. (c) The fluorophore is often attached to the end of the oligonucleotide. The technique can not only be used to label DNA, but also be combined with immunolabeling e.g., with functional antibody fractions. (d) When labeling a structure such as the DNA (d.1), often some binding partners are left unlabeled (d.2), and the linker (e.g., antibody) between the fluorophore and the target site (d.4), as well as the finite imaging precision (d.5), result in a deteriorated reconstruction (d.6). (e) Single molecule localization microscopy (SMLM) of nuclear DNA labeled with Hoechst [37]. Scale bar: 2 µm. The effect of limited labeling efficiency when imaging chromatin structure is clearly visible, with better image quality obtained when the number of detected fluorophore signals is increased from 30 to 2500 per µm².Due to abundancy, structural resolution could possibly be much higher when DNA specific dyes are used as opposed to labeling DNA bound proteins [38]. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA. Reprinted from [23] with permission from Wiley.

Figure 2

Imaging chromatin conformation. (a–c) Pairs of fluorescent dyes are used to study interactions such as binding between labeled proteins. Single molecule Förster resonance energy transfer (FRET) facilitates the real-time detection of chromatin conformational dynamics. (a) Left: A tetranucleosome structure is labeled with three dye pairs DA1, DA2, and DA3. Right: The chromatin fiber can be modeled as a stack of tetranucleosome (TN) units. The middle tetranucleosome carries the fluorescent labels, whose accessible volume is displayed. D donor, A acceptor labels, N nucleosomes. (b) Scheme of the total internal reflection fluorescence (TIRF) experiment to measure intra-array single molecule FRET. (c) Microscopic images showing FRET data of single chromatin arrays, scale bar: 5 µm. (d) Structural data from super-resolution microscopy can be combined with sequencing information (bottom) [91] to yield with higher confidence a model of the underlying structure. (a–c) © 2018 under the Creative Commons license [92] by Kilic et al. [83]. (d) Created with CSynth [91].