Background

Gene expression is thought to be regulated by the spatiotemporal organization of chromatin from the smallest length scale of individual nucleosomes (~10 nm) up to ~100 nm [1,2]. Even larger tertiary structures such as enhancer promoter contacts or topologically associated domains may exist that regulate gene expression [1,3]. The correlated movement between small chromatin structures and the large chromatin domains they are part of has been suggested as an important feature of nuclear phase condensates [4–6]. To understand these effects of chromatin structure and dynamics on gene regulation, imaging techniques are required that can characterize both the nanoscopic structure and the dynamics of chromatin in the larger context of chromatin domains they reside in.

Recently, two main breakthroughs facilitated research advances in this field: CRISPR/dCas9-based fluorescence labeling methods, to image specific sequences of chromatin, and super-resolution microscopy techniques such as photoactivated localization microscopy (PALM). By using programmable guide RNAs (gRNA), fluorophores can be targeted via CRISPR/dCas9 to specific sequences in the genome. Tens of fluorescent probes are required to be bound to a locus of interest to create a signal that is distinguishable from the background fluorescence of all other freely diffusing and searching probes [7]. To amplify the fluorescence signal of bound probes, repetitive RNA aptamers such as MS2 sequences have been attached to gRNAs that facilitate binding of multiple fluorophores. These labeling strategies facilitated conventional fluorescence timelapse imaging and yielded valuable insights into the slow and long-term dynamics of entire loci [7–9]. However, due to the optical diffraction limit, the structural characterization of smaller chromatin structures below ~250 nm has not been possible with conventional fluorescence microscopy. The development of PALM [10–12] enabled the tracking of single molecules in living cells (also referred to as single-particle tracking PALM, sptPALM) [13,14] and the acquisition of images of intracellular structures in fixed cells with ~20 nm resolution [15]. In PALM imaging, the spatiotemporal overlap of individual fluorophores is avoided by sparsely activating photoactivatable or photoswitchable fluorophores. In this way, the precise locations of individual fluorophores can be determined by Gaussian fitting of their intensity profile. By photoactivating and localizing many or all fluorophores over time, enough localizations are obtained to resolve structures below the optical diffraction limit in fixed cells or to link molecular trajectories to characterize their diffusion in live cells. CRISPR/dCas9-based DNA labeling has recently been applied in PALM to obtain structural information of chromatin compaction or condensation in chemically fixed cells or to characterize the dynamics of chromatin in live eukaryotic and prokaryotic cells [16]. However, until recently, it was not possible to simultaneously obtain such structural and dynamic information in living cells due to the motion of DNA during the long PALM data acquisition time. This motion spreads out the localizations of bound fluorophores along the trajectory of a locus and thus results in motion blurring.

Here, we present a protocol for our recently developed correlative conventional fluorescence and PALM imaging approach in living cells that overcomes these challenges [17]. This approach is based on labeling an intracellular structure or locus with a conventional fluorophore to track its location and motion during the entire PALM data acquisition time. Each structure or locus is, in addition, labeled with a spectrally distinct PALM-compatible fluorophore to record the single molecule localizations. The trajectory of the locus determined from the conventional fluorescence signal is then subtracted from the coordinates of its single molecule localizations to correct for motion blurring. As a result, high-resolution structural parameters such as the time-averaged size or the density of bound probes can be quantified to obtain new insights into the compaction of chromatin. In addition, the dynamics of the relative single molecule rearrangement or the motion of the entire locus can be related to its structural parameters, which has not been possible with existing techniques.

We demonstrate correlative conventional and PALM imaging using the well-characterized telomere sequences as a model system labeled via dCas9 and the MS2 coat proteins (MCP) that bind to a modified telomere-targeting gRNA scaffold. However, this imaging approach can be extended to single loci or other intracellular structures that can be labeled with a sufficient number of conventional and PALM-compatible fluorophores to create signals above background. Importantly, our presented data acquisition and analysis pipeline is a primary step to any downstream analysis to quantify structural or dynamic parameters. For instance, we demonstrate that determining the location and mobility of a locus relative to the single dCas9/MCP fluorophores classifies them more reliably as bound to a locus. The relative motion of single molecules compared to the entire locus furthermore reveals how small-scale chromatin rearrangements occur within the larger-scale chromatin movements. We also relate the compaction of telomeres to the local and global chromatin mobility to yield new insights. This protocol demonstrates that correlative conventional fluorescence and PALM imaging accurately identifies Cas9 molecules bound to a locus and yields quantitative dynamic and time-averaged structural information about specific genomic loci at the nanoscale in living cells. The versatility of this protocol makes it applicable to other organelles and enables other existing or future downstream analysis techniques to extract and correlate high-resolution structural features with dynamic parameters.