Accurate Sizing of Monodisperse Microbubble Suspensions by Optical Attenuation Spectroscopy

Objective Accurate sizing of ultrasound contrast agent microbubbles is crucial for understanding their behavior through a direct comparison between models and experimental data. However, a gold standard method for microbubble sizing has yet to be established. Methods We introduced optical attenuation spectroscopy (OAS) for the sizing of monodisperse microbubble suspensions. This method employs a spectrophotometer to obtain an optical attenuation spectrum, to which a Mie scattering model is fitted, yielding the modal bubble radius and polydispersity index. We compared OAS sizing with the most common methods: Coulter Counter, brightfield microscopy and fluorescence microscopy. Results OAS measurements were in excellent agreement (±1.5%) with fluorescence microscopy. In contrast, the Coulter Counter over-estimated the modal microbubble radius compared with OAS by 4% for 3.0 μm radius microbubbles, increasing up to 11% for 4.0 μm radius microbubbles. Brightfield microscopy systematically over-estimated the microbubble radius by 0.45 μm compared with fluorescence microscopy. Conclusion OAS has proven to be a versatile method for sizing monodisperse microbubble suspensions due to its convenient, label-free, and rapid acquisition within seconds.