Details

Autor(en) / Beteiligte

• VAN ELSLAND, D.M.
• BOS, E.
• PAWLAK, J.B.
• OVERKLEEFT, H.S.
• KOSTER, A.J.
• VAN KASTEREN, S.I.

Titel

Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling

Ist Teil von

• Journal of microscopy (Oxford), 2017-09, Vol.267 (3), p.309-317

Ort / Verlag

England: Wiley Subscription Services, Inc

Erscheinungsjahr

2017

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 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. Lay Description Fluorescence microscopy is a powerful imaging tool that can be employed to track, localize and monitor fluorescently labelled biomolecules within cellular systems. However, using this technique only fluorescently labelled biomolecules can be studied within a given sample, while pertinent information on their immediate cellular environment remains ‘in the dark’. Electron microscopy enables in depth observations at a nanometre‐scale resolution of precisely such environmental details, including the cytoskeleton, membranes and even macromolecular complexes. Therefore, electron microscopy is often employed after fluorescence microscopy to ultrastructurally characterize the cellular environment of fluorescently labelled biomolecules of interest. In order to translate the information derived from fluorescence‐based studies for electron microscopy analysis, fluorescently labelled biomolecules must be identified in a manner compatible with the latter. Traditionally, this is achieved using electron dense gold markers equipped with antibodies recognizing fluorescently labelled biomolecules. Alternatively, the area of interest is imaged with both the fluorescence and electron microscope, followed by superimposition of the resulting data sets. In order to compare and contrast the outcomes of these two strategies, we compared the subcellular localization of fluorescent labels with that of gold labels. Surprisingly, we found that the distribution of the gold label does not always correspond with the distribution of the fluorescent label. The present study experimentally interrogates and characterizes this phenomenon with broad implications for combining fluorescence and electron microscopy techniques.