Ultraplex microscopy: versatile highly-multiplexed molecular labeling and imaging across scale and resolution.

Area of Science:

• Cell Biology
• Microscopy Techniques
• Molecular Imaging

Background:

• Studying molecular organization in cells and tissues is hindered by inefficient multiplexed labeling and limited microscopy modality combinations.
• High spatial resolution is often required but difficult to achieve with current multiplexing and imaging methods.

Purpose of the Study:

• To introduce ultraplex microscopy, a novel technique that overcomes limitations in multiplexed molecular labeling and multi-modal imaging.
• To enhance resolution and expand multiplexing capacity for detailed cellular and tissue analysis.

Main Methods:

• Ultraplex microscopy integrates serial multiplexing, ultrathin sectioning, and reversible embedding.
• It allows labeling with antibodies, RNA probes, and tissue stains.
• Imaging is compatible with brightfield, epifluorescence, super-resolution, and electron microscopy without specialized reagents.

Main Results:

• Demonstrated applications in brain tissue, including molecular profiling of single cells and axonal boutons.
• Achieved high-resolution molecular colocalization.
• Successfully performed correlative imaging of fluorescent proteins with confocal and ultraplex microscopy.

Conclusions:

• Ultraplex microscopy circumvents incompatibilities between labeling and imaging techniques, enhancing resolution and multiplexing capacity.
• The technique is versatile and valuable for addressing complex experimental questions in various biological systems.
• It offers a powerful new approach for detailed molecular and spatial analysis in tissues.