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Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling
Author(s) -
VAN ELSLAND D.M.,
BOS E.,
PAWLAK J.B.,
OVERKLEEFT H.S.,
KOSTER A.J.,
VAN KASTEREN S.I.
Publication year - 2017
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/jmi.12567
Subject(s) - immunogold labelling , labelling , bioorthogonal chemistry , fluorescence , microscopy , context (archaeology) , electron microscope , fluorescence microscope , biophysics , biomolecule , chemistry , biology , biochemistry , optics , click chemistry , physics , paleontology , combinatorial chemistry
Summary Electron microscopy (EM) is traditionally employed as a follow‐up to fluorescence microscopy (FM) to resolve the cellular ultrastructures wherein fluorescently labelled biomolecules reside. In order to translate the information derived from FM studies to EM analysis, biomolecules of interest must be identified in a manner compatible with EM. Although fluorescent signals can serve this purpose when FM is combined with EM in correlative light and electron microscopy (CLEM), the traditional immunogold labelling remains commonly used in this context. In order to investigate how much these two strategies relate, we have directly compared the subcellular localization of on‐section fluorescence labelling with on‐section immunogold labelling. In addition to antibody labelling of LAMP‐1, bioorthogonal click labelling was used to localize soluble cysteine cathepsins or membrane‐associated sialylated glycans. We reveal and characterize the existence of inherent discrepancies between the fluorescence signal and the distribution of gold particles in particular in the case of membrane‐associated antigens.