Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling

Area of Science:

• Cell Biology
• Microscopy Techniques

Background:

• Fluorescence microscopy (FM) is often followed by electron microscopy (EM) to visualize cellular ultrastructure.
• Correlative light and electron microscopy (CLEM) bridges FM and EM, but requires compatible labeling methods.
• Traditional immunogold labeling is common in CLEM, alongside fluorescence-based approaches.

Purpose of the Study:

• To directly compare on-section fluorescence labeling with on-section immunogold labeling.
• To investigate the relationship between fluorescence signals and gold particle distribution in CLEM.
• To identify potential discrepancies in subcellular localization data obtained by these two methods.

Main Methods:

• Direct comparison of on-section fluorescence and immunogold labeling.
• Utilized antibody labeling for LAMP-1.
• Employed bioorthogonal click chemistry for soluble cysteine cathepsins and membrane-associated sialylated glycans.

Main Results:

• Identified inherent discrepancies between fluorescence signals and gold particle distribution.
• Observed significant differences, especially for membrane-associated antigens.
• Characterized the nature of these localization discrepancies.

Conclusions:

• Fluorescence and immunogold labeling methods can yield different subcellular localization results.
• Discrepancies are pronounced for membrane proteins and glycans, affecting CLEM interpretation.
• Careful consideration of labeling strategy is crucial for accurate ultrastructural localization in CLEM.