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© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

Post-embedding correlative light and electron microscopy (CLEM) has the advantage of high-precision registration and enables light and electron microscopy imaging of the same slice. However, its broad application has been hampered by the limited available fluorescent proteins (FPs) and a low signal-to-background ratio (SBR). Here, we developed a green photoswitchable FP, mEosEM-E with substantially high on/off contrast in EM samples embedded in Epon resin, which maximally preserves cellular structures but quenches the fluorescence of FPs. Taking advantage of the photoswitching property of mEosEM-E, the autofluorescence background from the resin was significantly reduced by a subtraction-based CLEM (sCLEM) method. Meanwhile, we identified a red fluorescent protein (RFP) mScarlet-H that exhibited higher brightness and SBR in resin than previously reported RFPs. With mEosEM-E and mScarlet-H, dual-colour post-Epon-embedding CLEM images with high SBR and no cross-talk signal were successfully performed to reveal the organization of nucleolar proteins. Moreover, a dissection of the influences of different EM sample preparation steps on the fluorescence preservation for several RFPs provides useful guidance for further probe development.

Details

Title
Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM
Author
Peng, Dingming 1 ; Li, Na 2 ; He, Wenting 3 ; Drasbek, Kim Ryun 4   VIAFID ORCID Logo  ; Xu, Tao 5 ; Zhang, Mingshu 1   VIAFID ORCID Logo  ; Xu, Pingyong 6   VIAFID ORCID Logo 

 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; [email protected] (D.P.); [email protected] (N.L.); [email protected] (W.H.); Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China 
 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; [email protected] (D.P.); [email protected] (N.L.); [email protected] (W.H.); Sino-Danish College, University of Chinese Academy of Sciences, Beijing 100101, China; Sino-Danish Center for Education and Research, Beijing 100039, China; [email protected]; Center of Functionally Integrative Neuroscience, Department of Clinical Medicine, Aarhus University, 8000 Aarhus, Denmark 
 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; [email protected] (D.P.); [email protected] (N.L.); [email protected] (W.H.) 
 Sino-Danish Center for Education and Research, Beijing 100039, China; [email protected]; Center of Functionally Integrative Neuroscience, Department of Clinical Medicine, Aarhus University, 8000 Aarhus, Denmark 
 Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China; National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 
 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; [email protected] (D.P.); [email protected] (N.L.); [email protected] (W.H.); Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China; National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 101408, China 
First page
1077
Publication year
2022
Publication date
2022
Publisher
MDPI AG
e-ISSN
20734409
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2649003123
Copyright
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.