Correlative super-resolution fluorescence and electron microscopy using conventional fluorescent proteins in vacuo

Area of Science:

• Microscopy
• Cell Biology
• Biophysics

Background:

• Advanced microscopy techniques like super-resolution light microscopy (SRLM), correlative light and electron microscopy (CLEM), and volume electron microscopy (vEM) are transforming biological sample analysis.
• Precisely correlating fluorescent proteins with cellular structures remains a challenge, often complicated by specimen manipulation between imaging modalities.

Purpose of the Study:

• To develop a novel, integrated microscopy approach for highly accurate correlation of fluorescent proteins to cellular structures.
• To overcome limitations of conventional section-based CLEM by minimizing specimen alteration and simplifying correlation.

Main Methods:

• Integration of a scanning electron microscope (SEM) with an optical microscope for simultaneous acquisition of localization microscopy and electron microscopy images.
• Imaging of YFP and GFP embedded in acrylic resin under partial vacuum to enhance blinking properties for in vacuo single molecule localization microscopy (SMLM).
• Acquisition of data from ultrathin sections to improve axial resolution and signal-to-noise ratio.

Main Results:

• Demonstrated enhanced blinking properties of YFP and GFP in acrylic resin under partial vacuum, enabling in vacuo SMLM.
• Achieved precise correlation of localization microscopy and electron microscopy data without specimen manipulation between systems.
• Showcased the technique's performance on vaccinia virus and diacylglycerol in cellular membranes, with potential for 3D correlation over large volumes.

Conclusions:

• The integrated microscopy approach provides super-accurate correlation of fluorescent proteins to cellular structures.
• This technique minimizes artifacts and simplifies correlation, advancing the understanding of biological samples at high resolution.
• Future expansion to arrayed sections promises unprecedented 3D correlative analysis over large volumes.