Chemiluminescence-Driven Tyramide Amplification for High-Sensitivity and Long-Term Immunofluorescence Imaging

Immunofluorescence (IF) staining serves as a fundamental technique for visualizing biomolecule localization. However, its sensitivity and effectiveness are often constrained by photobleaching, photodamage, and tissue autofluorescence, particularly in low-abundance targets or in long-term imaging. Here, we present ChemTIF, a chemiluminescence-triggered IF method that overcomes these intrinsic limitations by eliminating strong external light excitation. ChemTIF integrates horseradish peroxidase (HRP)-catalyzed tyramide signal amplification with luminol-based chemiluminescence, leveraging localized chemiluminescence resonance energy transfer to excite fluorophores at the target site. In contrast to conventional IF imaging that directly excites fluorophores with a strong laser, our method achieves up to 25-fold amplification in cells and 12-fold signal amplification in tissues under the same low-power illumination, while significantly reducing background noise and photodamage. In cellular and tissue models, ChemTIF demonstrated superior sensitivity, extended signal duration (up to 33 and 57 min for cells and tissues, respectively), and minimal autofluorescence, thereby outperforming the conventional IF technique (6-13 min for cells and tissues, respectively) in both static and dynamic imaging studies. Its compatibility with cyclic imaging further supports high-throughput applications with minimal photodamage for multiplex detection in tissues. Collectively, these capabilities position ChemTIF as a robust platform, enabling high-fidelity longitudinal imaging in spatial proteomics and biomedical research.