Characterization and calibration of the iQID digital autoradiography system for direct quantitative imaging of beta‐emitters in tissue samples

Background Autoradiography provides microscale mapping of radionuclide distributions, offering a promising approach to complement nuclear medicine imaging for small‐scale radiopharmaceutical therapy (RPT) research. However, while quantitative protocols for α‐emitters are well‐established, comparable methods for β‐emitters, such as the widely used theranostic radionuclide 177 Lu, remain underdeveloped. Bridging this gap is essential for correlating microscopic dose deposition with biological outcomes. Purpose The objective of this study was to characterize and calibrate the ionizing‐radiation Quantum Imaging Detector (iQID) digital autoradiography system specifically for 177 Lu. The work aimed to establish a validated methodology for converting raw count rates into absolute activity and to benchmark the system's quantitative accuracy against standard preclinical µSPECT/CT imaging. Methods The iQID system response was evaluated for key performance metrics, including spatial resolution, detection efficiency, background and minimum detectable activity, and depth dependence. Experimental characterization data were compared with Geant4 Monte Carlo simulations to verify the accuracy of radiation transport modeling. To establish a linearity profile, count rates were measured across an activity range of 0–300 Bq. Finally, cross‐modality benchmarking was performed using a custom stacked multi‐layer virtual water phantom to directly compare iQID performance with a calibrated preclinical µSPECT/CT system in a realistic measurement scenario. Results The iQID system exhibited a high linear response ( R 2 > 0.99) across the tested activity range. The effective spatial resolution for 177 Lu was determined to be around 43 µm. In the cross‐modality comparison, the iQID system achieved a total activity estimate of (0.194 ± 0.022) MBq, agreeing within 2% of the dispensed reference activity (0.197 ± 0.015) MBq. Crucially, the iQID system demonstrated superior quantitative accuracy for small‐scale features (0.8–2.5 mm diameters), successfully resolving activity concentrations in regions where µSPECT/CT performance was severely limited by partial volume effects. Conclusion This study establishes a validated framework for quantitative 177 Lu digital autoradiography. By resolving microscopic activity distributions that are imperceptible to macroscopic imaging, the iQID system provides the necessary resolution for accurate ex vivo activity quantification. This work lays the groundwork for detailed characterization of radiopharmaceutical biodistribution, enabling more precise correlations between localized uptake and tissue microenvironment features.