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.

mEosEM withstands osmium staining and Epon embedding for super-resolution CLEM.

Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO4) treatment for improved SR-CLEM.

Fast Super-Resolution Imaging Technique and Immediate Early Nanostructure Capturing by a Photoconvertible Fluorescent Protein.

Low temporal resolution and limited photocontrollable fluorescent protein probes have restricted the widespread application of single-molecule localization microscopy (SMLM). In the current study, we developed a new photoconvertible fluorescent protein (PCFP), pcStar, and quick single molecule-guided Bayesian localization microscopy (Quick-SIMBA). The combination of pcStar and Quick-SIMBA achieved the highest temporal resolution (0.1-0.25 s) with large field-of-view (76 × 9.4 µm2 -76 × 31.4 µm2) among the SMLM methods, which enabled the dynamic movements of the endoplasmic reticulum dense tubular matrix to be resolved. Moreover, pcStar extended the application of SMLM to imaging the immediate early nanostructures in Drosophila embryos and revealed a specific "parallel three-pillar" structure in the neuronal-glial cell junction, helping to elucidate glial cell "locking" and support of neurons during Drosophila embryogenesis.