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

Fu, Zhifei; Peng, Dingming; Zhang, Mingshu; Xue, Fudong; Zhang, Rui; He, Wenting; Xu, Tao; Xu, Pingyong

doi:10.1038/s41592-019-0613-6

Cited by 60 publications

(73 citation statements)

References 25 publications

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“…We first investigated whether or not monomeric green fluorescent proteins including mEGFP (monomeric EGFP with the A206K mutation) (λex max = 488 nm, λem max = 507 nm, pKa = 6.0) 10 , mWasabi (λex max = 493 nm, λem max = 509 nm, pKa = 6.5) 11 , mEosEM 8 , and CoGFP variant 0 isolated from Cavernularia obesa (CoGFPv0) (λex max = 498 nm, λem max = 507 nm, pKa = 6.0) 12 retain their fluorescence after osmium-staining. Cells expressing each fluorescent protein were fixed with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde at 4 °C for 1 h, and stained with 1% osmium tetroxide at 4 °C for 10 min.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, it has been reported that two monomeric fluorescent proteins, mKate2 tagged with seven amino acids (GGGGSGL) (mKate2-GGGGSGL) and mEosEM retain their fluorescence after osmium staining and Epon embedding 7 , 8 . mKate2 is a far-red fluorescent protein 9 .…”

Section: Introductionmentioning

confidence: 99%

“…mKate2 is a far-red fluorescent protein 9 . mEosEM is a photoconvertible fluorescent protein 8 . The discovery of these proteins suggests that there may be other fluorescent proteins suitable for in-resin CLEM of Epon-embedded samples.…”

Section: Introductionmentioning

confidence: 99%

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Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins

Tanida

Furuta

Yamaguchi

et al. 2020

Sci Rep

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In-resin CLEM of Epon embedded samples can greatly simplify the correlation of fluorescent images with electron micrographs. The usefulness of this technique is limited at present by the low number of fluorescent proteins that resist CLEM processing. Additionally, no study has reported the possibility of two-color in-resin CLEM of Epon embedded cells. In this study, we screened for monomeric green and red fluorescent proteins that resist CLEM processing. We identified mWasabi, CoGFP variant 0, and mCherry2; two green and one red fluorescent proteins as alternatives for in-resin CLEM. We expressed mitochondria-localized mCherry2 and histone H2B tagged with CoGFP variant 0 in cells. Green and red fluorescence was detected in 100 nm-thin sections of the Epon-embedded cells. In the same thin sections, we correlated the fluorescent signals to mitochondria and the nucleus using a scanning electron microscope. Similar results were obtained when endoplasmic reticulum-localized mCherry2 and histone H2B tagged with CoGFP variant 0 were expressed in the cells. Two-color in-resin CLEM of two cytoplasmic organelles, mitochondria and endoplasmic reticulum, was also achieved using mitochondria-localized mCherry2 and endoplasmic reticulum-localized mWasabi. In summary, we report three new fluorescent protein-alternatives suitable for in-resin CLEM of Epon-embedded samples, and achieved Epon-based two-color in-resin CLEM.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins

Tanida

Furuta

Yamaguchi

et al. 2020

Sci Rep

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“…Other fiducial particles with the same properties (Han et al, 2019; Kukulski et al, 2012; Takizawa et al, 2015) are equally suited for our approach. Moreover, other fluorescent probes (Fu et al, 2020; Hemelaar et al, 2017; Morrison et al, 2015; Paez-Segala et al, 2015; Tanida et al, 2020), sample preparation routines (Andrian et al, 2020; Brama et al, 2015; Hohn et al, 2015; C.J. Peddie et al, 2014), and resins (Zhou et al, 2017) developed to retain the available fluorescence signal after EM sample preparation can be adapted and used within the here described pipeline, using fiducials as correlation anchor.…”

Section: Discussionmentioning

confidence: 99%

Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes

Loginov

Fermie

Fokkema

et al. 2021

Preprint

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Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Directly linking molecular to nanoscale ultrastructural information is therefore crucial to understand cellular physiology. Volume or 3-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. Application of volume-CLEM is however hampered by limitations in throughput and 3D correlation efficiency. Addressing these limitations, we here describe a novel pipeline for volume-CLEM that provides high-precision (<100nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) data sets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated in a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume-EM, and obviates the need for post correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D.

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“…23,43,45 For example, recent developments in genetically encoded tags developed for CLEM/CSREM allow use of the conventional EM sample preparation route because of new osmium resistant photoactivatable fluorescent proteins, which enable super resolution microscopy on resin embedded sections. 44,46 Many (SR)-CLEM methods require fluorescent imaging prior to the EM sample preparation, 47 while others have adapted EM sample preparation protocols by reducing the amount of strong fixatives in order to preserve fluorescence. 39,43 However, the modifications on the embedding protocols oftentimes result in critical structural alterations and poorly preserved ultrastructure, which makes correlation between LM and EM images even more problematic.…”

Section: Introductionmentioning

confidence: 99%

Correlative cathodoluminescence electron microscopy bioimaging: towards single protein labelling with ultrastructural context

2020

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

Cited by 60 publications

References 25 publications

Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins

Two-color in-resin CLEM of Epon-embedded cells using osmium resistant green and red fluorescent proteins

Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes

Correlative cathodoluminescence electron microscopy bioimaging: towards single protein labelling with ultrastructural context

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