Whole-body tomographic fluorescence lifetime imaging of PD-L1 expression

Noninvasive quantification of immune markers such as programmed death ligand-1 (PD-L1) remains a major challenge because conventional molecular imaging methods cannot readily distinguish target-bound from nonspecifically retained probes within the tumor microenvironment. Here, we establish a framework for noninvasive quantification of PD-L1 expression in deep-seated hepatocellular carcinoma (HCC) using asymptotic time-domain (ATD) fluorescence lifetime tomography. In vivo time-domain fluorescence imaging was performed in mice bearing orthotopic HCC tumors following administration of a PD-L1-targeted near-infrared fluorescent probe $\left(\alpha \text{PDL}\mathrm{1-800}\right)$ . Multi-exponential analysis of time-domain fluorescence data was used to derive four amplitude-based metrics of PD-L1 expression. Quantitative in vivo measurements obtained from ATD tomography were validated against ex vivo PD-L1 expression measured by Western blotting. Among the evaluated metrics, a normalized parameter that accounts for inter-lifetime crosstalk demonstrated the strongest correlation with ex vivo PD-L1 expression (r² = 0.77). These findings establish a foundation for a noninvasive, nonionizing imaging approach to quantify and monitor receptor expression and support a future path for the longitudinal assessment of immune biomarkers in both preclinical studies and clinical settings.