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Identifying the scatterers and obtaining accurate estimates of ultrasonic scatterer sizes are beneficial adjuncts to characterize (diagnose) disease from ultrasonic backscatterer measurements. A new scatterer size estimation technique has been developed that is based on a 3D acoustic impedance map. Ultrasonic scatterer estimation techniques rely extensively on form factor functions to obtain estimates, and 3D impedance maps can be used to derive independently form factors. The 3D acoustic impedance map is derived from a 3D histological data set of tissue, and is independent of ultrasonically acquired data. A rat fibroadenoma and a mouse 4T1 mouse mammary tumor (MMT) were fixed (10% neutral-buffered formalin), embedded in paraffin, serially sectioned at 10 μm and 5 μm respectively, and stained with H&E for histologic evaluation. Each section was digitally photographed through the light microscope. Tissue structures in each section were assigned distinct acoustic impedance values. The images from serial sections were aligned to yield two 3D impedance data set. A Gaussian form factor was used to estimate scatterer size and acoustic concentration. The scatterer size estimates were compared to previous values that were obtained from ultrasonic backscatterer measurements (also using a Gaussian form factor). For both 3D impedance maps, the relative difference between the size estimates were below 10%. The optimization scheme was also conducted on two simulated medium and led to relative errors below 1% for the scatterer size. This approach demonstrates that the use of 3D impedance maps has significant potential for improving parametric imaging by evaluating form factor functions.