Unveiling the photophysical mechanistic mysteries of tetrazine-functionalized fluorogenic labels

Tetrazine-based fluorogenic labels are widely utilized in medical and biological studies, exhibiting substantial fluorescence enhancement (FE) following tetrazine degradation through bio-orthogonal reactions. However, the underlying mechanisms driving this fluorogenic response remain only partially resolved, particularly regarding the diminished FE efficiency in the deep-red and near-infrared (NIR) regions. This knowledge gap has impeded efforts to optimize these labels for extended emission wavelengths and improved FE ratios. This review offers a photophysical perspective, discussing the fluorescence quenching pathways (i.e., energy flows and charge separation) that regulate the fluorogenic properties exhibited in various types of tetrazine labels. Moreover, this work examines the emerging role of intramolecular rotations in certain tetrazine-based structures and the integration of additional quencher units. The proposed alternative quenching channel offers the potential to surpass traditional wavelength constraints while achieving improved FE. By examining these photophysical mechanisms, this review aims to advance the understanding of tetrazine-functionalized fluorogenic labels and provide guiding principles for their future design and practical applications.