Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Accurate protein localization is crucial for understanding cellular functions.
• Traditional fluorescence microscopy lacks subcellular context, while immuno-electron microscopy has limitations in antibody compatibility and antigen accessibility.
• Existing correlative methods struggle with resolution and precise protein mapping.

Purpose of the Study:

• To introduce and detail the nano-resolution fluorescence electron microscopy (nano-fEM) technique.
• To enable precise, nanoscale protein localization within cells.
• To overcome the limitations of existing protein mapping methodologies.

Main Methods:

• Nano-resolution fluorescence electron microscopy (nano-fEM) integrates photo-activated localization microscopy (PALM) and electron microscopy.
• Samples are fixed and embedded to preserve fluorescence, then sectioned into ultrathin slices.
• Sections are imaged using PALM for fluorescence and scanning electron microscopy for ultrastructure, with gold particles used for alignment.

Main Results:

• The nano-fEM technique achieves protein localization at the nanoscale.
• It overcomes the diffraction limit of light microscopy using PALM.
• The method provides precise subcellular context for protein distribution.

Conclusions:

• Nano-fEM offers a robust method for determining the subcellular localization of fluorescently tagged proteins at nanometer resolution.
• This technique significantly advances the ability to study protein function in its cellular context.
• The entire process can be completed in approximately one week.