Linking Adjuvant Immune Stimulation to Spatiotemporal Dynamics via Cross-Scale Photoacoustic Microscopy

Adjuvants can stimulate the innate immune system independently of vaccine antigens. Elucidating their spatiotemporal distribution across biological scales and correlating this distribution with immune activation are of considerable significance. A cross-scale photoacoustic/fluorescence dual-modality imaging system was developed to enable dynamic tracking of the adjuvant from the cellular to the organismal level. Single-cell imaging results revealed that macrophages exhibited differential activation after the uptake of different adjuvants. Notably, high-resolution photoacoustic microscopy (PAM) revealed size-dependent differences in the intravascular kinetics of adjuvant in zebrafish tail veins as well as their accumulation and clearance during circulation. In a tail amputation-induced inflammation model, fluorescence microscopy demonstrated that CpG oligodeoxynucleotides elicited a 5-fold enhancement in immune cell recruitment efficiency compared to controls, while nano/micron aluminum adjuvants induced 2-fold and 1.5-fold increases, respectively. The dual-modality photoacoustic/fluorescence system quantitatively established that these immune outcomes directly originate from the spatiotemporal dynamics of the adjuvant. By bridging single-cell phagocytic behavior with systemic biodistribution, this study deciphered a causal mechanism. The activation of the immune system by adjuvants results from the synergistic interaction of their chemical compositions and spatiotemporal distribution. This imaging-driven framework offers quantitative design principles for precision vaccine adjuvants.