Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biophysics

Background:

• Correlative light and electron microscopy (CLEM) is crucial for understanding cellular ultrastructure.
• Preserving cellular structure and fluorescence signals during sample preparation for CLEM is challenging.
• Super-resolution microscopy offers higher resolution but is difficult to combine with traditional EM sample preparation.

Purpose of the Study:

• To develop a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) in mammalian cells.
• To optimize sample preparation for preserving both structural integrity and fluorescence of standard proteins.
• To achieve nanoscale correlation between fluorescently labeled structures and ultrastructure in the same cell.

Main Methods:

• High-pressure freezing, freeze substitution, and resin embedding were optimized for fluorescence preservation.
• Standard fluorescent proteins (mGFP, mVenus, mRuby2) were used without special modifications.
• Single molecule localization microscopy (SMLM) was combined with transmission electron microscopy (TEM).

Main Results:

• Successful preservation of fluorescence and photo-switching of standard fluorescent proteins in cryo-fixed resin-embedded cells.
• Achieved a structural resolution of 40-50 nm in fluorescence images with ~17 nm localization accuracy.
• Enabled correlation of fluorescent labels to ultrastructure at the nanometer level.

Conclusions:

• The developed method allows for correlative super-resolution imaging and EM on the same cryo-fixed samples.
• This technique provides superior structural preservation compared to conventional methods.
• It facilitates detailed nanoscale investigations of cellular structures and their organization.