A method to validate quantitative high-frequency power doppler ultrasound with fluorescence in vivo video microscopy

Flow quantification with high-frequency (>20 MHz) power Doppler ultrasound can be performed objectively using the wall-filter selection curve (WFSC) method to select the cutoff velocity that yields a best-estimate color pixel density (CPD). An in vivo video microscopy system (IVVM) is combined with high-frequency power Doppler ultrasound to provide a method for validation of CPD measurements based on WFSCs in mouse testicular vessels. The ultrasound and IVVM systems are instrumented so that the mouse remains on the same imaging platform when switching between the two modalities. In vivo video microscopy provides gold-standard measurements of vascular diameter to validate power Doppler CPD estimates. Measurements in four image planes from three mice exhibit wide variation in the optimal cutoff velocity and indicate that a predetermined cutoff velocity setting can introduce significant errors in studies intended to quantify vascularity. Consistent with previously published flow-phantom data, in vivo WFSCs exhibited three characteristic regions and detectable plateaus. Selection of a cutoff velocity at the right end of the plateau yielded a CPD close to the gold-standard vascular volume fraction estimated using IVVM. An investigator can implement the WFSC method to help adapt cutoff velocity to current blood flow conditions and thereby improve the accuracy of power Doppler for quantitative microvascular imaging.