Přehled o publikaci

While the implementation of correlative super-resolution techniques has progressed in the last decade in mammalian models (Johnson et al., 2015; Kopek et al., 2012), the analysis of plant cellular ultrastructure with correlative techniques is often limited in plant systems due to limited tissue permeability, lack of antibodies, and trade-offs between good tissue preservation and accessibility for exogenous labelling. Another concern is sample thickness, as fluorescence microscopy benefits from thicker samples with more signal while transmission electron microscopy (TEM) requires ultra-thin sections between 60 – 100 nm. We present a method that combines super-resolution fluorescence microscopy (localization microscopy) with TEM, based on the use of click-iT chemistry for the efficient labelling of samples embedded in Lowicryl resin (Franek et al., 2024). Importantly, we show that the blinking behavior of fluorophores required for localization microscopy is maintained even after sample embedding in Lowicryl, indicating that the permeability of Lowicryl is sufficient for localization buffer access to the epitopes. Using this method, we studied nucleolar ultrastructure (provided by TEM) together with replication progression (provided by replication labelling coupled with click-iT chemistry) in the nanometer range. While the application of this method in a consecutive-section approach is preferable for high-quality localization microscopy, same-section CLEM can be also applied for exact localization of overlapping cellular features. This way, we have shown that the replication centers in the nucleolus are found in the vicinity of nucleolar fibrillar centers. Finally, we argue that applications of click-iT chemistry in a correlative setup will develop, as the detection step can be easily performed in samples already embedded in hydrophilic resins. We propose that other methods, such as chromatin contrasting with diaminobenzidine (DAB – Ou et al., 2017) could be added to workflows that use click-iT chemistry for the labelling of chromatin, with the added benefit of minimal background and high specificity.