Light-sheet fluorescence microscopy has revolutionized biology by visualizing dynamic cellular processes in three dimensions. However, light scattering in thick tissue and photobleaching of fluorescent reporters limit this method to studying thin or translucent specimens. In this study, we applied nondiffractive ultrasound beams in conjunction with a cross-amplitude modulation sequence and nonlinear acoustic reporters to enable fast and volumetric imaging of targeted biological functions. We reported volumetric imaging of tumor gene expression at the cubic centimeter scale using genetically encoded gas vesicles and localization microscopy of cerebral capillary networks using intravascular microbubble contrast agents. Nonlinear sound-sheet microscopy provides a ~64× acceleration in imaging speed, ~35× increase in imaged volume, and ~4× increase in classical imaging resolution compared with the state of the art in biomolecular ultrasound.

In contrast-enhanced echography, the simulation of nonlinear propagation of ultrasound through a population of oscillating microbubbles imposes a computational challenge. Also, the numerical complexity increases because each scatterer has individual properties. To address these problems, the Iterative Nonlinear Contrast Source (INCS) method has been extended to include a large population of nonlinearly responding microbubbles. The original INCS method solves the Westervelt equation in a four-dimensional spatiotemporal domain by generating increasingly accurate field corrections to iteratively update the acoustic pressure. The field corrections are computed by the convolution of a nonlinear contrast source with the Green's function of the linear background medium. Because the convolution integral allows a coarse discretization, INCS can efficiently deal with large-scale problems. To include a population of microbubbles, these are considered as individual contrast point sources with their own nonlinear response. The field corrections are computed as before, but now, in each iteration, the temporal signature of each contrast point source is computed by solving the bubble's Marmottant equation. Physically, each iteration adds an order of multiple scattering. Here, the performance of the extended INCS method and the significance of multiple scattering is demonstrated through various results from different configurations.

An in vivo range verification technology for proton beam cancer therapy, preferably in real-time and with submillimeter resolution, is desired to reduce the present uncertainty in dose localization. Acoustical imaging technologies exploiting possible local interactions between protons and microbubbles or nanodroplets might be an interesting option. Unfortunately, a theoretical model capable of characterising the acoustical field generated by an individual proton on nanometer and micrometer scales is still missing. In this work, such a model is presented. The proton acoustic field is generated by the adiabatic expansion of a region that is locally heated by a passing proton. To model the proton heat deposition, secondary electron production due to protons has been quantified using a semi-empirical model based on Rutherford's scattering theory, which reproduces experimentally obtained electronic stopping power values for protons in water within 10% over the full energy range. The electrons transfer energy into heat via electron-phonon coupling to atoms along the proton track. The resulting temperature increase is calculated using an inelastic thermal spike model. Heat deposition can be regarded as instantaneous, thus, stress confinement is ensured and acoustical initial conditions are set. The resulting thermoacoustic field in the nanometer and micrometer range from the single proton track is computed by solving the thermoacoustic wave equation using k-space Green's functions, yielding the characteristic amplitudes and frequencies present in the acoustic signal generated by a single proton in an aqueous medium. Wavefield expansion and asymptotic approximations are used to extend the spatial and temporal ranges of the proton acoustic field.