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Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

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Published on: December 3, 2012

Genetically encoded X-ray cellular imaging for nanoscale protein localization.

Huating Kong¹, Jichao Zhang¹, Jiang Li¹

¹Bioimaging Center, Shanghai Synchrotron Radiation Facility, Zhangjiang Laboratory, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China.

National Science Review

|October 25, 2021

Summary

Researchers developed genetically encoded X-ray-sensitive tags (GXET) using peroxidases for precise protein labeling in cells. This breakthrough enables ultra-high resolution imaging of biomolecular localization and interactions using X-ray microscopy.

Keywords:

3,3′-diaminobenzidine polymers cellular imaging genetically encoded X-ray tags nanoscale protein localization synchrotron-based X-ray microscopy

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Area of Science:

Cell biology
Microscopy
Biochemistry

Background:

Spatial resolution is crucial for understanding cellular processes.
X-ray microscopy (XRM) offers nanoscale resolution and deep tissue penetration.
Current biomolecule labeling methods are often complex and error-prone.

Purpose of the Study:

To develop a novel, genetically encoded labeling method for XRM.
To enable site-specific and efficient labeling of proteins in mammalian cells.
To achieve ultra-high resolution imaging of cellular structures and pathways.

Main Methods:

Engineered peroxidases were repurposed as genetically encoded X-ray-sensitive tags (GXET).
Fusion expression of peroxidases enabled site-specific labeling.
3,3'-diaminobenzidine (DAB) polymers were catalytically formed in situ.
XRM was used to visualize the DAB polymers with nanoscale resolution.

Main Results:

GXET successfully labeled proteins of interest in mammalian cells.
Catalytically formed DAB polymers were clearly visible under XRM.
Ultra-high resolution imaging of 30 nanometers was achieved.
Multicolor imaging was demonstrated using different peroxidase tags, leveraging XRM's energy resolution.

Conclusions:

GXET provides a powerful new tool for site-specific protein labeling in cells.
This method significantly advances nanoscopic imaging capabilities for biological research.
The developed technique facilitates high-resolution studies of biomolecular localization and interactions.