Abstract
In-resin CLEM of Epon embedded samples can greatly simplify the correlation of fluorescent images with electron micrographs. The usefulness of this technique is limited at present by the low number of fluorescent proteins that resist CLEM processing. Additionally, no study has reported the possibility of two-color in-resin CLEM of Epon embedded cells. In this study, we screened for monomeric green and red fluorescent proteins that resist CLEM processing. We identified mWasabi, CoGFP variant 0, and mCherry2; two green and one red fluorescent proteins as alternatives for in-resin CLEM. We expressed mitochondria-localized mCherry2 and histone H2B tagged with CoGFP variant 0 in cells. Green and red fluorescence was detected in 100 nm-thin sections of the Epon-embedded cells. In the same thin sections, we correlated the fluorescent signals to mitochondria and the nucleus using a scanning electron microscope. Similar results were obtained when endoplasmic reticulum-localized mCherry2 and histone H2B tagged with CoGFP variant 0 were expressed in the cells. Two-color in-resin CLEM of two cytoplasmic organelles, mitochondria and endoplasmic reticulum, was also achieved using mitochondria-localized mCherry2 and endoplasmic reticulum-localized mWasabi. In summary, we report three new fluorescent protein-alternatives suitable for in-resin CLEM of Epon-embedded samples, and achieved Epon-based two-color in-resin CLEM.
Highlights
Fluorescence microscopy with multi-color fluorescent proteins is an essential tool in the field of cell biology
Two color in‐resin Correlative light-electron microscopy (CLEM) of nucleus, mitochondria and endoplasmic reticulum in the Epon‐embedded cells was achieved using CoGFP variant 0 and mCherry[2]. Using these green and red fluorescent proteins, we investigated whether two color in-resin CLEM of nucleus and mitochondria in the Epon-embedded cells could be achieved
We investigated the possibility of using two-color in-resin CLEM for analysis of cytoplasmic organelles: endoplasmic reticulum and mitochondria
Summary
Fluorescence microscopy with multi-color fluorescent proteins is an essential tool in the field of cell biology. A proposed solution for these problems is to use Lowicryl HM20 (acrylate- and methacrylate-based) resins instead of Epon resins and high pressure freezing and freeze substitution techniques for in-resin CLEM with standard fluorescent proteins (mEGFP, mVenus, mRuby[2], and YFP). These techniques require special instruments and special r esins[3,4,5,6]. MEosEM is a photoconvertible fluorescent p rotein[8] The discovery of these proteins suggests that there may be other fluorescent proteins suitable for in-resin CLEM of Epon-embedded samples
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