Integrated super resolution fluorescence microscopy and transmission electron microscopy.

Area of Science:

• Microscopy
• Cell Biology
• Biophysics

Background:

• Correlative light and electron microscopy (CLEM) combines fluorescence microscopy (FM) and electron microscopy (EM) for multi-modal imaging.
• A key limitation in CLEM is the significant resolution difference between FM and EM.
• Current CLEM workflows often involve separate setups, risking sample damage and complicating region identification.

Purpose of the Study:

• To develop and present an integrated super-resolution FM and TEM system for enhanced CLEM.
• To reduce the resolution gap between FM and EM in CLEM.
• To improve correlation accuracy and streamline the CLEM workflow by avoiding sample transfer.

Main Methods:

• Integration of a wide-field super-resolution FM within a Transmission Electron Microscope (TEM).
• Utilized a rotating TEM sample holder for seamless switching between imaging modalities.
• Employed fiducial markers for accurate image overlay between FM and TEM datasets.
• Experimentally validated on Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin.

Main Results:

• Demonstrated successful overlay of TEM and FM images with an accuracy of 28 ± 11 nm.
• Achieved overlay accuracy close to the optical resolution limit of ~50 nm.
• Presented initial imaging experiments on labeled endothelial cells, showcasing the system's capability.

Conclusions:

• The integrated super-resolution CLEM system effectively bridges the resolution gap between FM and EM.
• The presented approach minimizes sample manipulation, reducing potential damage and improving workflow efficiency.
• This integrated system offers a promising advancement for high-resolution cellular ultrastructure analysis in correlative microscopy.