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Abstract The spatial organization and dynamics of a genome are central to gene regulation. While a comprehensive understanding of chromatin organization in the human nucleus has been achieved using fixed-cell methods, measuring the dynamics of specific genomic regions over extended periods in individual living cells remains challenging. Here, we present a robust and fully genetically encoded system for fluorescent labeling and long-term tracking of any accessible non-repetitive genomic locus in live human cells using fluorogenic and replenishable nanobody array fusions of theStaphylococcus aureusdCas9, and compact polycistronic single guide (sg)RNAs. First, we characterize the selectivity and photostability of our probes, enabling genome-wide visualization of chromatin dynamics at locally repetitive elements. Next, through multiplexed expression of 8–10 sgRNAs from polycistronic cassettes, we demonstrate efficient and sustained labeling of non-repetitive loci, enabling high-fidelity tracking of gene-proximal regions at exceptional spatial and temporal resolution. Finally, by correlating chromatin mobility with transcriptional activity at multiple genes, we find that local chromatin dynamics at 20 Hz are gene-specific and not necessarily dependent on transcription. Our approach is versatile, minimally invasive, and scalable, enabling multiplexed imaging of regulatory element dynamics involved in gene control, with broad applicability across diverse biological systems and disease contexts. 

Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin – a protein located in the focal adhesion complex – and actin in human osteosarcoma cells.