Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM

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.

Correlative Light, Electron Microscopy and Raman Spectroscopy Workflow To Detect and Observe Microplastic Interactions with Whole Jellyfish

“…While correlative microscopy with TEM providing a high-resolution validation of initially observed fluorescence signals has been highly utilized in other fields, very little work has been conducted to optimize this work flow for MP detection in tandem with Raman spectroscopy in whole marine organisms. , Additionally, it is often not possible to image the fluorescence of a sample after the necessary pre-processing steps for TEM due to the degradation of the molecules which give fluorescence (e.g., thermal degradation, pH degradation, or potential photobleaching during resin permeation and curing, and/or binding of heavy metal stains needed to improve TEM contrast) or to collect Raman spectra from highly fluorescent bulk samples without sacrificing signal intensity through the use of lower energy near infrared lasers. − Thus, our work focused on overcoming these analytical concerns for the first time for samples of whole marine organisms exposed to MPs. A protocol was developed which allowed for an initial insight into how interactions with C.…”

“… 26 , 27 Additionally, it is often not possible to image the fluorescence of a sample after the necessary pre-processing steps for TEM due to the degradation of the molecules which give fluorescence (e.g., thermal degradation, pH degradation, or potential photobleaching during resin permeation and curing, and/or binding of heavy metal stains needed to improve TEM contrast) or to collect Raman spectra from highly fluorescent bulk samples without sacrificing signal intensity through the use of lower energy near infrared lasers. 28 − 31 Thus, our work focused on overcoming these analytical concerns for the first time for samples of whole marine organisms exposed to MPs. A protocol was developed which allowed for an initial insight into how interactions with C. andromeda medusa may differ depending on the MP type, providing a platform for future research which can further probe such differences in these and other marine organisms.…”

Rapid in-EPON CLEM for everyone: Combining fast and efficient labeling of self-labeling enzyme tags with EM-resistant Janelia Fluor dyes

“…A recent approach to simplify post-embedding CLEM is to use fluorescent proteins which can, to some degree tolerate the quenching properties of conventional EM sample preparation protocols including osmium tetroxide staining and dehydration at room temperature. For this, special variants of the Eos-FP like mEosEM were engineered or standard fluorescent proteins like mWasabi, mKate2 or mScarlet-H were tested for their resistance to TEM sample preparation (Fu et al, 2020; Paez-Segala et al, 2015; Peng et al, 2022; Tanida et al, 2020). However, many of these proteins seem to retain only comparably weak fluorescence signals and low signal to background ratio after EPON embedding, and require protocols with reduced osmium tetroxide concentration (Peng et al, 2022).…”

“…For this, special variants of the Eos-FP like mEosEM were engineered or standard fluorescent proteins like mWasabi, mKate2 or mScarlet-H were tested for their resistance to TEM sample preparation (Fu et al, 2020; Paez-Segala et al, 2015; Peng et al, 2022; Tanida et al, 2020). However, many of these proteins seem to retain only comparably weak fluorescence signals and low signal to background ratio after EPON embedding, and require protocols with reduced osmium tetroxide concentration (Peng et al, 2022).…”

Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

“…One is the fluorescence quenching by the staining agents, which necessitates the use of an additional imaging modality (e.g. X-ray computational tomography) for localization of the area of interest [30][31][32] . Another one is the need to use microtomy to prepare a starting plane for the FIB milling.…”