Multiplex Protein Imaging through PACIFIC: Photoactive Immunofluorescence with Iterative Cleavage

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Multiplex protein imaging is crucial for deep phenotyping and spatial biology.
• Current methods present trade-offs, necessitating innovative imaging toolkits.
• There is a persistent need for advanced techniques to analyze cellular heterogeneity.

Purpose of the Study:

• To introduce PACIFIC (photoactive immunofluorescence with iterative cleavage), a new multiplex protein imaging modality.
• To demonstrate PACIFIC's capability for iterative multiplexing using photocleavable fluorophores and spin-column purification.
• To showcase PACIFIC's utility in analyzing cellular heterogeneity in FFPE samples and fixed cells.

Main Methods:

• Development of PACIFIC utilizing photocleavable fluorophores for antibody labeling.
• Implementation of one-step spin-column purification for antibody-fluorophore conjugates.
• Integration of PACIFIC with live-cell tracking for functional studies.

Main Results:

• PACIFIC enables iterative multiplexing without specialized instruments, DNA encoding, or harsh chemical treatments.
• The method successfully resolves cellular heterogeneity in both formalin-fixed paraffin-embedded (FFPE) samples and fixed cells.
• Integration with live-cell tracking identified phosphor-p70S6K as a key regulator of U87 cell mobility.

Conclusions:

• PACIFIC offers a cost-effective, flexible, and compatible approach to multiplex protein imaging.
• The technology democratizes deep phenotyping capabilities for researchers using traditional immunofluorescence platforms.
• PACIFIC advances the toolkit for spatial biology and cellular heterogeneity studies.