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Correlated light and electron microscopy: ultrastructure lights up!

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Abstract

Microscopy has gone hand in hand with the study of living systems since van Leeuwenhoek observed living microorganisms and cells in 1674 using his light microscope. A spectrum of dyes and probes now enable the localization of molecules of interest within living cells by fluorescence microscopy. With electron microscopy (EM), cellular ultrastructure has been revealed. Bridging these two modalities, correlated light microscopy and EM (CLEM) opens new avenues. Studies of protein dynamics with fluorescent proteins (FPs), which leave the investigator 'in the dark' concerning cellular context, can be followed by EM examination. Rare events can be preselected at the light microscopy level before EM analysis. Ongoing development—including of dedicated probes, integrated microscopes, large-scale and three-dimensional EM and super-resolution fluorescence microscopy—now paves the way for broad CLEM implementation in biology.

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Figure 1: Matching scales.
Figure 2: Examples of CLEM with distinct approaches.
Figure 3: CLEM procedures and considerations.
Figure 4: Commercial integrated CLEM microscopes.
Figure 5: Cx43 as a 'guinea pig' in CLEM probe development.
Figure 6: Sequential versus integrated CLEM.

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Acknowledgements

We thank C.J. Peddie and L.M. Collinson for providing Figure 2d,e and our departmental members for feedback. We acknowledge financial support for our CLEM work from the Netherlands Organization for Scientific Research (ZonMW91111006; “Microscopy Valley” STW12718 and STW12714; NWO175-010-2009-023), the NanoNextNL innovation programme (09A.04) and a Marie Curie International Reintegration Grant within the 7th European Community Framework Program.

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Correspondence to Jacob P Hoogenboom or Ben N G Giepmans.

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J.P.H. is cofounder and shareholder at Delmic BV, one of the manufacturers of integrated microscopes mentioned in this work.

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de Boer, P., Hoogenboom, J. & Giepmans, B. Correlated light and electron microscopy: ultrastructure lights up!. Nat Methods 12, 503–513 (2015). https://doi.org/10.1038/nmeth.3400

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