In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

Michal Franek (), Lenka Koptašíková, Jíří Mikšátko, David Liebl, Eliška Macíčková, Jakub Pospíšil, Milan Esner, Martina Dvořáčková () and Jíří Fajkus
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Michal Franek: Masaryk University
Lenka Koptašíková: Charles University, Faculty of Science, Biology Section, Imaging Methods Core Facility at BIOCEV
Jíří Mikšátko: Charles University, Faculty of Science, Biology Section, Imaging Methods Core Facility at BIOCEV
David Liebl: Charles University, Faculty of Science, Biology Section, Imaging Methods Core Facility at BIOCEV
Eliška Macíčková: Charles University, Faculty of Science, Biology Section, Imaging Methods Core Facility at BIOCEV
Jakub Pospíšil: Central European Institute of Technology Masaryk University (CEITEC MU)
Milan Esner: Central European Institute of Technology Masaryk University (CEITEC MU)
Martina Dvořáčková: Masaryk University
Jíří Fajkus: Masaryk University

Nature Communications, 2024, vol. 15, issue 1, 1-13

Abstract: Abstract Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre 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 sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a 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 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Date: 2024
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DOI: 10.1038/s41467-024-46324-6

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