Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM.

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.