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Real-time GHz Ultrasonic Imaging of Nematodes at Microscopic Resolution

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This work reports high-frequency ultrasonic imaging to visualize and study microscopic pests such as nematodes. This imaging modality using ultrasound instead of light enables visualizing nematodes even under the soil. This GHz technology uses a compact 128 x 128-pixel array of transducers that allow imaging at a sampling rate of up to 12 fps. The images can be viewed in real-time and further processed to measure the worms’ spatial dimensions such as length, width, as well as velocity. Here, we show that the ultrasonic imaging approach can be a powerful tool to visualize and detect microscopic pests such as nematodes and study their behavior in their natural habitat.
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1594 Microsc. Microanal. 28 (Suppl 1), 2022
doi:10.1017/S1431927622006389 © Microscopy Society of America 2022
Real-time GHz Ultrasonic Imaging of Nematodes at Microscopic Resolution
Anuj Baskota1*, Justin Kuo1 and Amit Lal1
1. Geegah Inc., Ithaca, NY, United States
* Corresponding author: anuj@geegah.com
Nematodes are worm-like multi-cellular animals that are known to cause damage to the roots, stems, and
foliage of plants. They are responsible for billions of dollars’ worth of damage per year in the crop
industry worldwide [1]. Plant-pest management should accurately detect and identify nematodes to carry
out necessary action in the farm before any damages occur. Several identification methods of nematodes
are based on biochemical, morphological, and molecular features [2]. Most of these require extraction
and processing of soil samples, chemical analysis, Polymerase Chain Reaction (PCR), enzymes
extraction, and other complex biochemical processing [3-6]. These processes can be time-consuming,
laborious, and requires specialized facilities/laboratories to perform analysis. Similarly, several imaging
techniques previously used to visualize and study nematodes include visible light, thermal,
hyperspectral, and atomic force microscopes [7,8]. Most of these microscopy techniques are expensive
and bulky. In addition, several limitations among these techniques arise due to the impermeability of
light in soil. Ultrasound imaging has been frequently used in medical diagnostic processes where the
frequency used ranges from 2 15 MHz. In this work, we propose high frequency (1.8 GHz) ultrasonic
imaging as a potential tool to visualize and study microscopic pests such as nematodes. In previous
work, we demonstrated the use of this GHz ultrasonic imager array to sense nematodes in air, water, and
soil [9]. This work further analyzes the reflected echo data to extract spatial features of two different
nematode species (Steinernema carpocapsae and Steinernema feltiae). The ultrasonic imaging modality
enables measurements of acoustic impedance and phase shifts allowing potential differentiation between
various nematode species. Furthermore, the high frequency ultrasonic imaging approach can be a
powerful tool to visualize, as well as detect microscopic pests at a very high sampling rate.
We use a compact 128 x 128-pixel array of Aluminum Nitride (AlN) transducers that allow imaging at a
sampling rate of up to 12 fps enabling real-time visualization of nematodes motion with a spatial
resolution of each pixel as 50 µm (Figure 1A). The necessary circuitry is integrated within the pixel
using a 130 nm CMOS process that results in a low-power and low-cost chip. As the wavelength is
inversely proportional to frequency, imaging at GHz frequency allows for high imaging resolution (~ 4.5
µm at 1.85 GHz). When voltage is applied to each transducer, the wave travels through the silicon,
reflects on the backside, and is received back by the transducer (Figure 1 B). The intensity of wave
reflecting depends on the acoustic impedance of material located on the silicon backside. The reflected
echo amplitude for 128 x 128 pixels can be plotted to obtain an image.
The experimental setup has an optical microscope camera (Hayear HY-2307) mounted on top, and the
ultrasonic imager placed right below it. The camera is synchronized with the imager’s data acquisition to
verify that the ultrasonic images show nematodes. Similarly, the optical images were used to measure
nematode dimensions and moving velocity and compare them with the values obtained through the
acoustic images. Nematodes Steinernema carpocapsae and Steinernema feltiae (obtained from BioBest
Sustainable Crop Management) were washed thoroughly with water to separate any impurities/food
particles. Few nematodes from both species were then dispensed on the imager surface and left to dry
separately. As the water dried up, both ultrasonic and optical reading was taken until all the nematodes
https://doi.org/10.1017/S1431927622006389 Published online by Cambridge University Press
Microsc. Microanal. 28 (Suppl 1), 2022 1595
completely dried up. ImageJ [10] and Tracker[11] were used to measure the spatial dimensions of the
nematodes and their moving speed, respectively.
The presence of nematode was verified by its shape and motion in the ultrasonic images. The nematodes
can be clearly seen crawling on the imager’s surface (Figure 2). For both the species, the measured
length from ultrasonic images is in the same range as obtained from optical (two paired parametric t-test
p > 0.5 for both species) as shown in Figure 3A. The measured velocities for both species was obtained
to be in the range ~0.05 0.5 mm/s, as shown in Figure 3B. The measured moving velocities also
correspond to values reported from literature [12] for nematode species of similar dimensions.
Figure 1 A) Four pixels (50 µm * 50 µm each) with integrated CMOS T/R circuits. B) Schematic
showing ultrasonic imaging of nematodes.
Figure 2. Series of ultrasonic images (reflected echo) shows nematode (S. feltiae) moving on the imager
surface. The white scale bar corresponds to 300 µm.
Figure 3 A) Length distribution of S. carpocapsae (n = 24) and S. feltiae (n = 86) measured optically and
ultrasonically. B) Velocity distribution measured from ultrasonic images for S. carpocapsae and S.
feltiae swimming in a thin water layer on silicon surface (n = 8 for both species).
https://doi.org/10.1017/S1431927622006389 Published online by Cambridge University Press
1596 Microsc. Microanal. 28 (Suppl 1), 2022
References:
[1] S. Koenning and K. Wrather, Plant Health Progress, Nov. 2010.
[2] Y. Seesao, M. Gay, S. Merlin, E. Viscogliosi, C. M. Aliouat-Denis, and C. Audebert, Journal of
Microbiological Methods, vol. 138, pp. 3749, Jul. 2017
[3] S. Mattiucci, G. Nascetti, L. Bullini, P. Orecchia, and L. Paggi, Parasitology, vol. 93, no. 2, pp. 383
387, Oct. 1986
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Ultrasonics Symposium (IUS), Sep. 2021
[10] C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, Nature Methods, vol. 9, no. 7, pp. 671675,
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[11] Tracker, Physlets.org, 2022.
[12] S. Jung, Physics of Fluids, vol. 22, no. 3, p. 031903, Mar. 2010.
https://doi.org/10.1017/S1431927622006389 Published online by Cambridge University Press
... However, the spatial resolution (0.5 -2mm) provided by this frequency range is insufficient for smaller organisms to capture their fine structural details. On the other hand, GHz frequencies offer significantly higher resolution of ∼0.5 m to 50 , which have been previously used to study cells [9], [10], soil nematodes [11], thin chitosan films [12], and metamaterial lenses [13]. Therefore, GHz Ultrasonic Imaging has the potential to resolve mechanical properties of C. elegans at a higher resolution. ...
... First the propagation of ultrasound through the silicon thickness and nematode thickness is modeled by the transfer matrix , and the propagation of ultrasound through the silicon thickness with an air backing is modeled by the transfer matrix , as below, where the silicon, cuticle, and muscle layers are represented by ABCD matrices , , and , respectively, which take the generic form where is the acoustic impedance of the layer, ℓ is the thickness of the layer, and is the propagation constant of the layer material = + β, where is the attenuation constant of the material and is the wave number of the material and is defined as 2 / , where is the ultrasonic wavelength in the material [21]. ] (11) To model the pressure at the transmit plane of the silicon layer, the source impedance matrix [ ] is multiplied by parallel matrices , and , used to model the acoustic propagation stack placed in shunt with the source impedance, where is the acoustic impedance of silicon. ...
... The overall packaging of the GHz ultrasonic imager is compact and handheld, with a silicon surface as the sensing region ( Figure 1A). More details on the circuitry and fabrication of the GHz imager, with another application of imaging nematodes, are described in our previous works [3,4]. Ultrasonic pulses generated by the transmitting pixels are transmitted and reflected off the backside of the silicon. ...
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  • S Koenning
  • K Wrather
S. Koenning and K. Wrather, Plant Health Progress, Nov. 2010.
  • Y Seesao
  • M Gay
  • S Merlin
  • E Viscogliosi
  • C M Aliouat-Denis
  • C Audebert
Y. Seesao, M. Gay, S. Merlin, E. Viscogliosi, C. M. Aliouat-Denis, and C. Audebert, Journal of Microbiological Methods, vol. 138, pp. 37-49, Jul. 2017
  • S Mattiucci
  • G Nascetti
  • L Bullini
  • P Orecchia
  • L Paggi
S. Mattiucci, G. Nascetti, L. Bullini, P. Orecchia, and L. Paggi, Parasitology, vol. 93, no. 2, pp. 383-387, Oct. 1986