For some time Riaud had been working on an idea for a new medical device, inspired by evidence that cancerous cells exhibit different physical properties than healthy cells. In particular, cancerous cells are softer—easier to deform—than their healthy counterparts. This suggests that measuring cell softness could be a way to diagnose cancer and monitor its progression, and maybe other diseases as well.
A colored scanning electron micrograph of a human T lymphocyte cell. Evidence suggests that cancerous T cells are softer than their healthy counterparts. Credit: National Institute of Allergy and Infectious Disease.
Riaud was a post-doc in Valerie Taly’s research group at Paris Descartes University (Paris V) when he started thinking about this possibility. After reading up on the subject, he realized that none of the existing techniques for measuring cell softness could work in a clinical setting. They either required elaborate setups with high-speed video cameras or cells samples that didn’t reflect the reality of biological specimens, or both. So, Riaud and colleagues from Paris V and his current institution, Fudan University in China, proposed a new technique.
The technique, published in the American Physical Society’s journal Physical Review Applied, brings together two processes—resistive pulse sensing (RPS) and acoustophoresis. RPS is a well-established method for determining the size of a cell or particle. The particle travels through a narrow channel filled with a liquid that conducts electricity. The particle’s presence changes the electrical conductivity of the channel, and does so by an amount that depends on its volume. By measuring the change in conductivity, researchers can infer the size of the particle.
Illustration of an RPS system in which particles (red) are suspended in a weakly conducting fluid (blue). Particles flow through a narrow channel and are sensed electrically by electrodes (yellow) placed on either side of the channel. Credit: Mymathematix (CC BY-SA 4.0).
Acoustophoresis is intimidating word, but really it’s just the combination of “acoustics” (sound) and “phoresis” (migration); it’s the process of using sound to move objects. That might sound a little strange at first—unless you count yelling, we don’t typically try to move things with sound. But sound waves are physical vibrations (aka pressure waves) that travel through molecules, so they push on every object in their path. Because sound is a wave, everything usually ends up back in place. However, if the wave is strong enough, an object in its path can experience a steady push. This is called acoustic radiation pressure.
The size of the force depends on the frequency of the sound wave, the speed of sound, and the physical properties of the object in its way. By exploiting these relationships, you can design a standing wave —a wave “frozen” in place—capable of trapping or moving objects. For example, by orienting a standing wave against gravity, researchers have been able to levitate water droplets, small particles, insects, and even small fish using sound, usually ultrasound.
Left: This image shows the acoustic levitation of small objects (expanded polystyrene particles). Sound waves are produced by a source, shown at the top of the image, and reflect off of a concave reflector to create a standing wave. Right: Illustration showing the simulated standing wave pattern. Credit: M. Andrade/University of São Paulo.
Similarly, you can design a standing wave that sorts small floating objects into different “swimming lanes” of a channel according to their physical properties. You can then measure how much a particle is deflected by a certain acoustical power, or you can measure the acoustical power necessary to deflect a particle by a certain amount.
Riaud’s basic idea was to sort cells into groups using this technique, and then measure their sizes with RPS. How does that relate to softness? Well, using a mathematical model, he showed that a particle’s deviation depends on the acoustical power as well as the particle’s size, density, and softness. Therefore, if you know (or can measure) the acoustics, deviation, size, and density of a particle, it’s fairly straightforward to calculate its softness.
The idea came together quickly, Riaud says, but a proof-of-concept experiment and analysis took much longer. A six-month timeline turned into more than three years. “The key reason it took so long is that, as physicists, we often think at a very simplified and abstract level: ‘Cells are just particles, they all share the same radius,’” he explains. “Using actual cells is totally different: they die, they clump together, they break into debris. It took some time to adapt.”
The time-consuming proof-of-concept experiment involved two electronic chips, one for acoustophoresis and one for RPS. The sample, a liquid mixture containing tiny polystyrene spheres and human T lymphocyte cells, first entered a sorting channel. The particles were subjected to varying acoustical
powers and deflected to an outlet channel. The outlet channel fed into an RPS channel on the second chip, that detected the particle sizes.
“When I started to study these data, I found no correlation between the sorting power and the detection events, which was very frustrating and disappointing,” Riaud says. A few months later, while on his honeymoon, inspiration struck in the form of a shuttle boat ride. “I came to realize that like this boat, the cells must have been slowly traveling between the sorting and detection section. Still on [my honeymoon], I crafted a mathematical model of the cell dynamics, implemented it on the computer, and everything nicely fell into place.”
|A couple watching the sunset in Rio from a boat. Credit: Micaela Parente on Unsplash.|
After accounting for the time-delay and spending some additional quality time with the data, Riaud and the team showed that they could clearly distinguish spheres from cells by softness in their experiment. Even in the unexpected cases where spheres and cells overlapped in either size or softness, by knowing both properties the researchers could clearly distinguish between the two.
“[W]e are at the very beginning of a process that could enable physicians to measure not only the size of each cell, but also the weight, softness, polarizability, etc., in the hope that it helps in screening diseases” says Riaud. Such single-cell analysis is an exciting possibility and the teams plans to continue working toward this goal. They are currently studying how fast sound travels through cells and what the speed reveals about cell structure. Eventually, they’d like to do the same with even smaller objects like bacteria and viruses. This could dramatically improve our understanding of diseases, but “will be very challenging,” Riaud notes. Perhaps that means it’s time to start planning that second honeymoon?
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