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Understanding the pressure dependence of the nonlinear behavior ofultrasonically excited phospholipid (PL)-stabilized NBs is important foroptimizing US exposure parameters for implementations of contrast enhancedultrasound, critical to molecular imaging. The viscoelastic properties of theshell can be controlled by introduction of membrane additives, such aspropylene glycol as a membrane softener or glycerol as a membrane stiffener. Wereport for the first time, the production of high yield NBs with narrowdispersity and different shell properties. Through precise control over sizeand shell structure, we show how these shell components interact with thephospholipid membrane, change its structure, affect their viscoelasticproperties, and consequently change their acoustic response. A two-photonmicroscopy technique through a polarity-sensitive fluorescent dye, C-laurdan,was utilized to gain insights on the effect of membrane additives to themembrane structure. We report how the shell stiffness of NBs affects thepressure threshold (Pt) for the sudden amplification in the scattered acousticsignal from NBs. For narrow size NBs with 200 nm mean size, we find Pt to bebetween 123-245 kPa for the NBs with the most flexible membrane as assessedusing C-Laurdan, 465-588 kPa for the NBs with intermediate stiffness and588-710 kPa for the NBs with stiff membranes. Numerical simulations of the NBdynamics are in good agreement with the experimental observations confirmingthe dependence of acoustic response to shell properties, thereby substantiatingfurther the development in engineering the shell of UCAs. The viscoelasticdependent threshold behavior can be utilized for significantly and selectivelyenhancing the diagnostic and therapeutic ultrasound applications of potentnarrow size NBs.

High yield, shell-stabilized, narrow-sized C3F8 nanobubbles with  different shell properties and precisely controllable response to acoustic  excitations: experimental observations and numerical simulations