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    Area of Science:

    • Biophysics
    • Biomedical Engineering
    • Acoustics

    Background:

    • Genetically encoded gas vesicles (GVs) are protein nanostructures that scatter sound, enabling deep tissue cellular imaging.
    • GVs serve as a platform for biomolecular engineering, functioning as acoustic reporter genes and biosensors.
    • Cross amplitude modulation (xAM) is a sensitive and specific non-destructive ultrasound imaging method for GVs in vivo.

    Purpose of the Study:

    • To present advanced xAM theory and imaging principles for enhanced biomolecular ultrasound.
    • To detail analytical expressions, experimental observations, and noise characteristics of xAM beams.
    • To introduce beamforming methods for improving xAM contrast-to-noise ratio.

    Main Methods:

    • Derivation of analytical expressions for X-wave beam width and lobe distances.
    • Experimental validation of nondiffractive xAM beams and secondary lobe modulation.
    • Analysis of xAM image noise characteristics and development of beamforming techniques.

    Main Results:

    • Provided analytical expressions for xAM beam parameters.
    • Experimentally confirmed nondiffractive xAM beams and demonstrated secondary lobe level modulation.
    • Characterized xAM image noise as incoherent, enabling sensitivity enhancement via frame averaging.
    • Developed a beamforming formalism to improve contrast-to-noise ratio without sacrificing framerate.

    Conclusions:

    • The advancement of Biomolecular Ultrasound relies on the parallel development of genetically encoded probes and sophisticated imaging techniques like xAM.
    • xAM imaging principles and their 3D extensions, such as nonlinear sound-sheet microscopy, are crucial for the future of deep tissue imaging.
    • Optimized xAM methods offer enhanced sensitivity and contrast for ultrasound imaging of genetically encoded nanostructures.