Metrology-Guided High-Intensity Focused Ultrasound Reveals Spatially Confined Thermal Effects in Colorectal Cancer Cell Lines

Objective: Accurate assessment of biological effects in high-intensity focused ultrasound (HIFU) research is often limited by the lack of standardized calibration protocols for in vitro applications. This study established a metrology-guided calibration framework to evaluate the spatially confined thermal and biological effects of a Sonic Concepts H-102 HIFU transducer.Materials and Methods: Ultrasonic thermal doses were calculated using the Sapareto--Dewey model, and an automated 3D system was developed for precise temperature characterization. Biological effects of thermal doses (TD 60, TD 120, and TD 240 min) were investigated in HT-29 and HCT-116 colorectal cancer cell lines. Temperature elevation, cell viability (MTT assay), and apoptosis-related responses (TALI image cytometry and JC-1 assay) were compared between focal and adjacent wells.Results: The HIFU transducer achieved precise focal heating (50$degree$C - 52$degree$C) while avoiding significant thermal spread to adjacent regions (\>2 mm). HT-29 cells exhibited significant biological responses at focal sites with minimal effects in adjacent wells (p\<0.0001). HCT-116 cells demonstrated higher thermal sensitivity with less distinct focal confinement (p values ranged from \<0.001 to \>0.05), indicating that biological responses to indirect HIFU-related stress may vary according to intrinsic cell line characteristics.Conclusions: This metrology-guided platform provides a reliable, reproducible framework for standardized in vitro HIFU applications while revealing cell-line-specific differences in thermal sensitivity.