PT  - JOURNAL ARTICLE
AU  - Dingming Peng
AU  - Na Li
AU  - Wenting He
AU  - Kim Ryun Drasbek
AU  - Tao Xu
AU  - Mingshu Zhang
AU  - Pingyong Xu
TI  - Improved fluorescent proteins for dual-color post-embedding CLEM
AID  - 10.1101/2022.02.16.480633
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.02.16.480633
4099  - http://biorxiv.org/content/early/2022/02/16/2022.02.16.480633.short
4100  - http://biorxiv.org/content/early/2022/02/16/2022.02.16.480633.full
AB  - 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 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) mScharlet-H that exhibited higher brightness and SBR in resin than previously reported RFPs. With mEosEM-E and mScharlet-H, dual-color 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.Competing Interest StatementThe authors have declared no competing interest.