Contact-less, non-resonant and high-frequency ultrasonic technique: Towards a universal tool for plant leaf study

Plant-based measurements are recognized as key methods to obtain insightful data in the field. In general, they are labor-intensive and expensive. In this context, Non-Contact Resonant Ultrasonic Spectroscopy technique (NC-RUS) emerged as a powerful alternative that enabled plant water status determination in a non-destructive, non-invasive and rapid way. However, NC-RUS is not applicable to all plant species as it depends on the possibility to excite and sense thickness resonances in the leaves. In this work, we propose and test an ultrasonic technique that can be used in all leaves, regardless of the appearance of thickness resonances. This technique is based on the contactless measurement of through transmitted airborne ultrasonic pulses in the leaves at high-frequencies and in the absence of thickness resonances, to obtain the leaf ultrasonic velocity (vair). It benefits from the facts that: i) at sufficiently high frequencies (typically around 1 MHz) all leaves are non-resonant (so the technique can be applied to both resonant and non-resonant leaves), ii) the use of high-frequencies allows a greater time resolution and a further miniaturization, making possible to apply the technique to small and irregular leaves. Three different signal processing techniques were used to determine the time it takes to the ultrasonic pulse to cross the leaves (time-of-flight) from the measured signals. Two of them operate in time domain: cross-correlation, and edge detection, while the third one makes use of the Fast Fourier Transform (FFT) and operates in the frequency domain: phase-slope. If leaf thickness is also measured, ultrasound velocity can then be worked out. As ultrasound velocity is determined by density and elastic modulus, it is then closely related to water content and turgor pressure. Obtained ultrasound velocities were first validated by comparing them with those obtained by well-established and standard ultrasonic methods: water immersion transmission (vwater) and NC-RUS (vres). The conclusions of this comparison permitted us to propose a novel methodology that combines the three signal processing techniques used to improve robustness and accuracy for the measurement of ultrasound velocity in plant leaves. It is of interest to note that a bias towards higher values of vair compared to vres was observed. This behavior is considered the consequence of the different influence of the leaf layered structure in these two measurements, so this feature can be further used for leaf structure analysis.