A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags

Abstract

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

“…Here, were the structural preservation and labeling intensity satisfactory (Figure S3A,B) and sometimes quite good, though specimen contrast was lower than with cocktail UA/GA/OsO4. Such GA‐free samples from cells grown on (gridded) sapphire discs avoid the problem of GA‐induced autofluorescence and could, therefore be useful for certain kinds of complementary fluorescence microscopy and EM analyses . Notably, the majority of antibodies so far tested with our cryo‐based pre‐embedding approach was compatible with FS‐cocktails containing up to 0.05% OsO4 (in total: 11 out of 16).…”

A 4D view on insulin secretory granule turnover in the β‐cell

“…CLEM revealed that the ultrastructure of those complex objects resembled the classical morphology of MGBs, with several spherical electron dense particles enclosed within a large vesicle . Corroborating these findings, CLEM of labelled SOFIA mouse β‐cells revealed that starting 3 days after their biogenesis, more SGs were found within MGBs …”

“…The specific characteristics of the SNAP system allow for the analysis of the labelled SGs using different approaches including live‐cell imaging, microscopy of fixed samples, fluorimetry or immuno‐isolation (Figure ). Recently, we developed a protocol for correlative light and electron microscopy (CLEM) enabling the evaluation of labelled and unlabelled SGs in the same sample . By now, we successfully targeted insulin SGs of insulinoma cell lines and mice with the SNAP‐tag .…”

“…In this way, the number of insulin SGs of any given age can be easily assessed by calculating the ratio of Ins‐SNAP labelled SGs, as detected by FLM, relative to the total number of SGs visualized by EM. We have shown that combination of structured illumination microscopy and transmission electron microscopy allows for the precise localization of age‐distinct insulin SGs in Tokuyasu sections of SOFIA (Study OF Insulin Ageing) mouse β‐cells . The SOFIA mouse model contains a knock‐in of insulin‐SNAP into the Ins2 locus .…”

“…Accordingly, SGs have a mean half‐life of 4.6 days, which is consistent with previous estimates based on radiolabelling experiments . Since fluorescent SNAP substrates contain organic fluorophores they are not as sensitive to strong chemical fixation, heavy metal contrasting and embedding in epoxy resins for EM as self‐fluorescent proteins . This surprising phenomenon is compatible with superior fixation methods such as high pressure freezing (HPF), which preserves the ultrastructure of the sample in a state close to nature .…”