Nonlinear ultrasound imaging of nanoscale acoustic biomolecules

Abstract

Ultrasound imaging is widely used to probe the mechanical structure of tissues and visualize blood flow. However, the ability of ultrasound to observe specific molecular and cellular signals is limited. Recently, a unique class of gas-filled protein nanostructures called gas vesicles (GVs) was introduced as nanoscale ($250 nm) contrast agents for ultrasound, accompanied by the possibilities of genetic engineering, imaging of targets outside the vasculature and monitoring of cellular signals such as gene expression. These possibilities would be aided by methods to discriminate GV-generated ultrasound signals from anatomical background. Here, we show that the nonlinear response of engineered GVs to acoustic pressure enables selective imaging of these nanostructures using a tailored amplitude modulation strategy. Finite element modeling predicted a strongly nonlinear mechanical deformation and acoustic response to ultrasound in engineered GVs. This response was confirmed with ultrasound measurements in the range of 10 to 25 MHz. An amplitude modulation pulse sequence based on this nonlinear response allows engineered GVs to be distinguished from linear scatterers and other GV types with a contrast ratio greater than 11.5 dB. We demonstrate the effectiveness of this nonlinear imaging strategy in vitro, in cellulo, and in vivo. Published by AIP Publishing. [http://dx

Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI

“…Furthermore, we found that GVs’ unique physical properties enable them to produce harmonic ultrasound signals useful for contrast specificity in vitro and in vivo 9 , 10 . We also showed that GVs from different species can be imaged in multiplex based on their differential responses to acoustic pressure, and that conditional GV clustering leads to contrast enhancement, allowing them to be used as molecular sensors 8 .…”

“…Second harmonic imaging is operated as an option via the VSX GUI. Note that we have recently developed an amplitude modulation strategy that can also be used for highly selective non-linear imaging of native and engineered GVs 9 .…”

“…For ultrasound, unmodified Halo GVs can be used directly in ultrasound imaging to obtain non-linear signals 8 , 10 . Ana GVs are the system of choice if one wishes to genetically tune the properties of GVs for multiplexing, multimodal imaging and targeting applications 9 , 14 . Mega GVs produce lower echogenicity under ultrasound compared to Ana and Halo GVs (Figure S1, a-d), but have a higher critical collapse pressure that may make them useful for multiplexing.…”

“…Ana and Halo cultures natively produce ample GVs after a few weeks of growth 8 , 9 , 14 . Ana cultures additionally require a controlled gaseous environment and illumination for optimal growth (Figure 2a-b).…”

“…Buoyant cells are then lysed using hyper-osmotic shock for Ana and hypo-osmotic shock for Halo. Subsequently, centrifugally assisted floatation is used to isolate GVs from the cell lysate to yield a concentrated, milky-white solution of GVs in the buffer of choice 8 , 9 , 14 (Figure 2g-j). Heterologous production of Mega GVs in E. coli is accomplished by expression from a plasmid encoding a Mega GV gene, followed by detergent-mediated lysis 11 .…”