Collagen I is widely used as a natural component of biomaterials for both tissue engineering and regenerative medicine applications. The physical and biological properties of fibrillar collagens are strongly tied to variations in collagen fiber microstructure. The goal of this study was to develop the use of high-frequency quantitative ultrasound to assess collagen microstructure within three-dimensional (3D) hydrogels noninvasively and nondestructively. The integrated backscatter coefficient (IBC) was employed as a quantitative ultrasound parameter to detect, image, and quantify spatial variations in collagen fiber density and diameter. Collagen fiber microstructure was varied by fabricating hydrogels with different collagen concentrations or polymerization temperatures. IBC values were computed from measurements of the backscattered radio-frequency ultrasound signals collected using a single-element transducer (38-MHz center frequency, 13-47 MHz bandwidth). The IBC increased linearly with increasing collagen concentration and decreasing polymerization temperature. Parametric 3D images of the IBC were generated to visualize and quantify regional variations in collagen microstructure throughout the volume of hydrogels fabricated in standard tissue culture plates. IBC parametric images of corresponding cell-embedded collagen gels showed cell accumulation within regions having elevated collagen IBC values. The capability of this ultrasound technique to noninvasively detect and quantify spatial differences in collagen microstructure offers a valuable tool to monitor the structural properties of collagen scaffolds during fabrication, to detect functional differences in collagen microstructure, and to guide fundamental research on the interactions of cells and collagen matrices.

Beamforming includes a variety of spatial filtering techniques that may be used for determining sound source locations from near-field sensor array recordings. For this scenario, beamforming resolution depends on the acoustic frequency, array geometry, and target location. Random scattering in the medium between the source and the array may degrade beamforming resolution with higher frequencies being more susceptible to degradation. The performance of frequency-sum (FS) beamforming for reducing such sensitivity to mild scattering while increasing resolution is reported here. FS beamforming was used with a data-dependent [minimum variance (MV)] or data-independent (delay-and-sum, DAS) weight vector to produce higher frequency information from lower frequency signal components via a quadratic product of complex signal amplitudes. The current findings and comparisons are based on simulations and passive cavitation imaging experiments using 3 MHz and 6 MHz emissions recorded by a 128-element linear array. FS beamforming results are compared to conventional DAS and MV beamforming using four metrics: point spread function (PSF) size, axial and lateral contrast, and computation time. FS beamforming produces a smaller PSF than conventional DAS beamforming with less computation time than MV beamforming in free space and mild scattering environments. However, it may fail when multiple unknown sound sources are present.