Epon Post Embedding Correlative Light and Electron Microscopy.

Area of Science:

• Cell Biology
• Microscopy Techniques

Background:

• Correlative light and electron microscopy (CLEM) integrates fluorescence microscopy (FM) and electron microscopy (EM) for cellular analysis.
• Traditional CLEM often involves a compromise between fluorescence signal and ultrastructure preservation.
• Epon resin offers superior ultrastructure preservation compared to other embedding media.

Purpose of the Study:

• To develop and detail a protocol for Epon post-embedding CLEM.
• To achieve simultaneous preservation of fluorescence and ultrastructure in CLEM.
• To improve cell identification and image registration in CLEM.

Main Methods:

• Utilizing mEosEM, a fluorescent protein demonstrated to withstand osmium tetroxide fixation and Epon embedding.
• Implementing detailed step-by-step procedures for EM sample preparation, FM imaging, EM imaging, and image alignment.
• Refining methods for identifying the same cell across FM and EM imaging modalities and improving image registration.

Main Results:

• Successful Epon post-embedding CLEM achieved for the first time, maintaining both fluorescence and ultrastructure.
• Demonstrated the viability of mEosEM under osmium tetroxide fixation and Epon embedding conditions.
• Improved procedures for accurate correlation of FM and EM data.

Conclusions:

• The developed protocol enables simultaneous high-resolution ultrastructural and fluorescence imaging.
• This method overcomes previous limitations in CLEM, offering a balanced approach to imaging modalities.
• The protocol is designed for easy implementation in standard electron microscopy facilities.