Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron...

Area of Science:

• Cell Biology
• Microscopy
• Biochemistry

Background:

• Advanced microscopy techniques like confocal fluorescence microscopy (FM), super-resolution microscopy (SRM), and transmission electron microscopy (TEM) are crucial in cell biology.
• Correlative light and electron microscopy (CLEM) integrates live-cell dynamics with ultrastructural details.
• Existing genetically encoded markers are limited for CLEM applications requiring compatibility across multiple imaging modalities.

Purpose of the Study:

• To introduce tetramethylrhodamine (TMR) as a novel, multimodal marker for CLEM.
• To demonstrate TMR's utility in combining FM, SRM, and TEM data.
• To establish TMR as a versatile tool for advanced cell imaging.

Main Methods:

• TMR was coupled to ligands for genetically encoded self-labelling enzyme tags (HaloTag, SNAP-tag, CLIP-tag).
• TMR's fluorescent properties were utilized for FM and SRM imaging.
• TMR's photooxidizing capability for diaminobenzidine (DAB) was assessed for TEM visualization.

Main Results:

• TMR successfully functioned as a fluorochrome in FM and SRM with various enzyme tags.
• TMR demonstrated efficient photooxidation of DAB into an osmiophilic polymer, visible in TEM.
• The TMR-based system proved effective for CLEM across multiple imaging techniques in mammalian cells.

Conclusions:

• Tetramethylrhodamine (TMR) is a versatile multimodal marker suitable for FM, SRM, and TEM.
• TMR facilitates correlative imaging by bridging live-cell dynamics with ultrastructural details.
• This TMR-based approach enhances CLEM capabilities for detailed cell biology research.