One for All, All for One: A Close Look at In-Resin Fluorescence Protocols for CLEM

Area of Science:

• Microscopy
• Biotechnology
• Cell Biology

Background:

• Correlative light and electron microscopy (CLEM) requires sample preparation balancing fluorescence light microscopy (LM) and electron microscopy (EM) needs.
• Traditional EM sample prep uses heavy metal staining that often quenches fluorescence, hindering CLEM.
• In-resin fluorescence protocols enable fluorescence preservation during EM sample preparation.

Purpose of the Study:

• To critically review fundamental aspects of resin embedding for in-resin fluorescence CLEM.
• To discuss workflows, advantages, and disadvantages of current in-resin fluorescence approaches.
• To highlight future developments for advanced in-resin CLEM.

Main Methods:

• Review of specimen fixation, contrasting agents, and embedding resins for CLEM.
• Analysis of physics and chemistry for preserving fluorescence and antigenicity.
• Evaluation of methods for optimizing EM contrast (transmission EM, scanning EM, volume EM) alongside fluorescence.

Main Results:

• Mild heavy metal staining preserves fluorescence in resin-embedded specimens, a breakthrough for CLEM.
• In-resin fluorescence protocols offer high-precision localization of fluorescent signals in EM data.
• Compromises between fluorescence preservation and EM contrast (especially for vEM) remain a challenge.

Conclusions:

• In-resin fluorescence protocols represent a significant advancement for high-throughput CLEM.
• Careful consideration of fixation, contrasting, and resin choice is vital for successful in-resin CLEM.
• Future developments promise enhanced capabilities for integrated LM-EM approaches.