Click-EM for imaging metabolically tagged nonprotein biomolecules

Abstract

Electron microscopy (EM) has long been the main technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce “Click-EM,” a labeling technique for correlative light microscopy and EM imaging of non-protein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal “click chemistry” ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of Click-EM in imaging metabolically tagged DNA, RNA, and lipids in cultured cells and neurons, and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells

“…4A). ChromEM staining does not require genetically modified cells, overexpression of tagged histone fusions, or incorporation of nucleotide analogs (75), all of which could perturb DNA structure and function. As such, ChromEMT provides a facile and universal method to compare the structure of genomic DNA in different kingdoms of life.…”

High‐performance serial block‐face SEM of nonconductive biological samples enabled by focal gas injection‐based charge compensation

“…Cultured cells and tissues were prepared as previously described using a combination of glutaraldehyde fixation, ferrocyanide reduced osmium tetroxide post-fixation, thiocarbohydrazide and osmium tetroxide liganding, followed by en bloc uranyl acetate and lead aspartate staining (Deerinck et al , 2010, Ngo et al , 2016, Williams et al , 2011). Cultured cells were also stained for DNA using click-chemistry as previously described (Ngo et al ., 2016).…”

Three decades of listeriology through the prism of technological advances

“…Fluorescence super resolution techniques benefits from enhanced labeling methods allowing minimally invasive in situ visualisation of molecules without affecting their properties (Plamont et al, 2016;Sahl et al, 2017). Alternative probes smaller than antibodies are being developed such as nanobodies, aptamers and other non-protein biomolecules (Ngo et al, 2016;Sahl et al, 2017). Current imaging techniques also reveals phenotypic heterogeneity of microbial and cellular populations that can be further studied by systems biology approaches (Bumann, 2015;Kreibich & Hardt, 2015).…”