Research Divisions
Research Programs
Research Progress
Supporting System
Technology Transfer
Laboratory Safety & Security
Chinese Academy of
Sciences Protein
Science core facility
    Home > Research > Research Progress

Chinese research team has developed the first photo-controllable fluorescent protein for Epon based post-embedding super-resolution correlative light-electron microscopy

Author: Update time: 2019-10-11

Protein molecules assemble into protein machinery at specific locations in the cell to perform biological functions. Therefore, it is of vital importance to study the precise localization of these molecules to reveal the assembly and molecular mechanisms of protein machinery. Electron microscopy (EM) achieves sub-nanometer spatial resolution and is an indispensable research tool in the life sciences. However, locating a specific protein in an EM image is challenging. Immunoelectron microscopy had been commonly used for this purpose by taking advantage of an antigen-antibody reaction, however, the labeling efficiency is quite low and the it is often limited by the availability and specificity of the antibody. Correlative light and electron microscopy (CLEM) is a powerful tool that combines the light microscopy (LM) for locating target molecules with high labeling efficiency and specificity, and EM for providing the ultrastructural of the cellular context. The recently emerged super-resolution CLEM (SR-CLEM) improves LM resolution beyond the diffraction limit and narrows the resolution gap between LM and EM.
One of the main challenges faced by CLEM is that osmium tetroxide (OsO4) fixation and Epon embedding for maintaining cellular ultrastructure and contrast and ensuring sectioning property during EM sample preparation inevitably quench fluorescence of fluorescent proteins (FPs). The pre-embedding CLEM method images the whole cell by LM before the fluorescence is destroyed by the subsequent EM sample preparation. However, in these cases, precise image registration is not feasible due to the deformation of sample after EM sample preparation and unmatched Z-axial resolution between LM and EM. Therefore, it would be of great benefit to develop a fluorescent protein that can retain fluorescence after OsO4-fixation and Epon-embedding.
In a recent study conducted by the joint team of Dr. XU Tao and Dr. XU Pingyong from Institute of Biophysics, Chinese Academy of Sciences, the researchers developed the first photo-converted fluorescent protein mEosEM that remained fluorescence after routine EM preparation. The protein has photoswitchable property that are suitable for single molecule localization microscopy (PALM/STORM). By using mEosEM and optimizing the imaging acquisition method and the single molecule localization algorithm in ultrathin sections, Epon-embedding-based SR-CLEM using the same-section approach has been achieved for the first time, which greatly promoted the field of SR-CLEM.
The developed FP and SR-CLEM method can well preserve the subcellular structure in EM, and also retain the localization accuracy of PALM. Intracellular mitochondria and nuclear membrane were successfully localized and correlated using this technique. The development is expected to bring a wide range of applications in biology.

Figure 1 Epon-embedding-based SR-CLEM of mitochondrial and nuclear lamina A using the same-section approach

(Imaged by Dr. Xu Pingyong's group)

This work has been published online ahead of printing in Nature Methods on Oct 14th, 2019 tilted "mEosEM withstands osmium staining and Epon embedding for super-resolution CLEM”.


Article link:


Contact: Pingyong Xu
Institute of biophysics, Chinese Academy of Sciences
Beijing100101, China
Tel: (86)-10-64888808
Email: pyxu

(Reported by Dr. Xu Pingyong's group)


Print Text Size: A A A   Close


Institute of Biophysics, CAS    Address: 15 Datun Road, Chaoyang District, Beijing, 100101, China
Tel:8610-64889872    Fax: 8610-64871293    E-mail: