|Entry||Database: EMDB / ID: 4472|
|Title||Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy|
|Map data||Low-magnification tomogram for correlation with cryoSOFI data.|
|Sample||XC (rous sarcoma) cell transiently expressing Lifeact-Dendra2:|
|Method||electron tomography / cryo EM|
|Authors||Moser F / Prazak V / Mordhorst V / Andrade AM / Baker LA / Hagen C / Grunewald K / Kaufmann R|
|Citation||Journal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019|
Title: Cryo-SOFI enabling low-dose super-resolution correlative light and electron cryo-microscopy.
Authors: Felipe Moser / Vojtěch Pražák / Valerie Mordhorst / Débora M Andrade / Lindsay A Baker / Christoph Hagen / Kay Grünewald / Rainer Kaufmann
Abstract: Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context ...Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.
|Date||Deposition: Dec 16, 2018 / Header (metadata) release: Feb 27, 2019 / Map release: Feb 27, 2019 / Last update: Mar 20, 2019|
Downloads & links
|File||emd_4472.map.gz (map file in CCP4 format, 742896 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 46.2 Å|
CCP4 map header:
-Entire XC (rous sarcoma) cell transiently expressing Lifeact-Dendra2
|Entire||Name: XC (rous sarcoma) cell transiently expressing Lifeact-Dendra2|
Number of components: 1
-Component #1: cellular-component, XC (rous sarcoma) cell transiently expressing...
|Cellular-component||Name: XC (rous sarcoma) cell transiently expressing Lifeact-Dendra2|
Recombinant expression: No
|Source||Species: Rattus (rat)|
|Source (natural)||Organ or tissue: muscle|
|Specimen||Specimen state: cell / Method: cryo EM|
|Sample solution||Buffer solution: DMEM 10% fetal bofine serum / pH: 7.4|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: OTHER|
-Electron microscopy imaging
Model: Tecnai Polara / Image courtesy: FEI Company
|Imaging||Microscope: FEI POLARA 300|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 3 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 50000.0 X (nominal) / Cs: 2 mm / Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Processing||Method: electron tomography / Number of sections: 40|
|3D reconstruction||Algorithm: BACK PROJECTION / Software: eTomo|
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