In-section Click-iT detection and super-resolution CLEM: Shedding light on nucleolar ultrastructure and S-phase progression in plants.

Abstract

Correlative light and electron microscopy (CLEM) is an essential tool that allows for localisation of a particular target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometer scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of serial sections by both, single-molecule localization microscopy and transmission electron microscopy for correlative analysis. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows a dramatic improvement in fluorophore blinking behavior relative to the whole-mount approaches. Here we used commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as FASCIATA1 and NUCLEOLIN1 mutants and applied our on-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Plant phenotyping relevance

植物組織向けの超解像・電子顕微鏡CLEM撮像ワークフローを開発・実証しており、核小体構造やS期進行という植物状態の取得が中心的な方法論的貢献である。

Code and data availability

The supplied blocks describe a CLEM/SMLM workflow on Arabidopsis root sections, but contain no public phenotype/trait datasets, image deposits, author analysis code, or trained models. Analysis used third-party tools (ThunderSTORM, NanoJ-SQUIRREL, SMAP, IMOD, Scipy) without any authors' public repository or deposit URL

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