Sonic snares: Trapping microorganisms for deeper insights into swimming behavior and ciliary dynamics

Microswimmers are small-scale organisms (1) or synthetic particles (2) that possess the ability to propel themselves through fluids. They can exhibit a variety of propulsion mechanisms, including the coordinated beating of cilia or flagella (3) or the use of self-generated forces (4). Exploring these propulsion mechanisms provides a deeper understanding of how biological systems and engineered microscale objects can move and navigate their environments. However, freely swimming microorganisms can be challenging to control and image with sufficient resolution to study their locomotion. In this issue of PNAS, Cui et al. (5) present an innovative label-free acoustofluidic device which enables the confinement of individual cilia-driven cells in a controlled space. This cutting-edge methodology offered the temporal and spatial resolution to characterize three-dimensional body motion and cilia waveform of the unicellular alga Chlamydomonas reinhardtii, with no constraints in body rotation, swimming characteristics, and cellular phenotype expression. This groundbreaking technique opens up new possibilities for studying the intricate swimming behavior and ciliary dynamics of microorganisms without interfering with their natural behavior.

C. reinhardtii (6) has been widely employed for research in different areas, ranging from photosynthetic functions (7) and cell cycle regulation (8) to human ciliary diseases (9). The prominence of C. reinhardtii as a model organism can be attributed to several key factors, including the ability to synchronize its cell cycle and the examination of ciliary motion. Cilia play a crucial role in various physiological processes in the human body, including the propulsion of fluid flow in airways, cerebral ventricles, and the oviduct. Understanding the structure and function of cilia is essential for comprehending these mechanisms and their implications for human health. Gaining insights into this relationship could have important implications for addressing pathologies associated with ciliary dysfunction, such as primary ciliary dyskinesia, chronic otitis, chronic obstructive pulmonary disease, and infertility. By studying C. reinhardtii and its ciliary motion, researchers can uncover valuable information that may contribute to the development of strategies to diagnose and treat these conditions.

Over the past few decades, progress in manipulation methods has led to the emergence of trapping approaches that can confine and separate biologically active particles (10). These approaches have played a crucial role in advancing microbiology, medicine, and biophysics research, encompassing various scales, from tiny vesicles to large multicellular clusters, with noteworthy achievements (11, 12). One cutting-edge technology that has been extensively adopted in the field is microfluidics (13). Microfluidic devices provide several advantages for cell trapping and isolation. They excel in handling small volumes, typically in the range of nanoliters to microliters, which is particularly advantageous when working with limited sample quantities. Additionally, microfluidics offers versatility in fabricating channels that are commensurate with the size of cells, enabling precise control over complex microenvironments, making them pivotal tools for the thorough investigation of cellular behaviors in a more realistic and physiologically relevant context. Furthermore, microfluidics facilitates high-throughput analysis, allowing researchers to study individual cells or small populations with enhanced resolution. This capability is particularly valuable for tracking individual cell responses over time, thus providing a deeper understanding of cellular heterogeneity and the underlying factors that contribute to cellular activity and function.

The process of capturing and manipulating small particles or cells in a liquid medium is commonly referred to as hydrodynamic trapping. This technique utilizes physical barriers to selectively separate and confine specific cells or particles from the surrounding fluid. Hydrodynamic trapping can be classified into two main categories: contact-based and contactless methods. Contact-based approaches encompass both vertical trapping and lateral trapping techniques. Vertical trapping occurs when cells or particles are captured in a downward direction relative to the fluid flow, which is often achieved using microwells or micropits where the cells or particles are immobilized (14). On the other hand, lateral trapping refers to the capture of cells or particles in a sideways manner relative to the fluid flow. This is typically accomplished using structures such as jail bars, obstacles, or traps that divert the flow and trap the cells or particles laterally (15). In contrast, contactless approaches in hydrodynamic trapping do not involve direct physical contact between the cells and the trapping region. Instead, external fields are utilized to exert forces on the cells for trapping. Examples of contactless approaches include magnetic trapping, where magnetic fields are used to control the movement and capture of magnetically labeled cells or particles. Optical tweezers employ focused laser beams to trap and manipulate cells based on the principle of radiation pressure. Dielectrophoretic trapping utilizes electric fields to induce movement and confinement of cells or particles based on their dielectric properties. Acoustic trapping involves the use of acoustic waves to create trapping forces and immobilize cells or particles at specific locations within the fluidic system.

“In summary, the development of single-cell manipulation techniques, such as the hybrid BAW/SAW acoustic tweezers device created by Cui et al., represents a significant breakthrough in the field of microswimmer analysis and offers new opportunities for investigating ciliary/flagellar dynamics in response to external perturbations, including mechanical, chemical, or optical stimuli.”

In their study, Cui et al. (5) employed a modified version of acoustic trapping, commonly referred to as acoustic tweezers, to confine swimming microorganisms. Acoustic tweezers represent a promising and emerging technology that allows for the precise manipulation of particles by utilizing the interaction of sound waves with various media, including solids, liquids, and gases (16). The principles behind acoustic tweezers involve the generation of acoustic waves that exert forces on cells and particles, enabling their spatial and temporal positioning. Acoustic tweezers offer unique advantages compared to other manipulation techniques, particularly in terms of the length scale, spanning from 10⁻⁷ to 10⁻² m, and it has been demonstrated to be safe for a range of diagnostic applications. By optimizing the operational parameters, such as frequency and intensity of the acoustic waves, researchers have been able to avoid potential damage to cells and small animal models, as reported in the case of red blood cells and Zebrafish embryos (17). Therefore, acoustic tweezers offer an alternative noninvasive approach to manipulate biological samples, providing advantages such as compatibility with nonoptical samples, reduced risk of damage to samples, and the ability to exert stronger forces on larger particles or cells. These advantages make acoustic tweezers a promising tool for various biomedical and biophysical applications.

Acoustic tweezers can be categorized into three main types: standing-wave tweezers, traveling-wave tweezers, and acoustic-streaming tweezers. Standing-wave tweezers manipulate particles or fluids by utilizing the acoustic radiation force generated by standing waves. These tweezers create regions of constructive and destructive interference, leading to the formation of pressure nodes and antinodes within the fluid medium. The particles or cells experience forces that depend on their physical properties, such as size, shape, and acoustic properties. There are two subtypes of standing-wave tweezers: surface acoustic wave (SAW) tweezers and bulk acoustic wave (BAW) tweezers. SAW-based devices are often integrated into microfluidic systems due to their compact size and compatibility with miniaturized platforms (18). In a previous study (19), it was observed that traditional SAW devices, when operated at low input power, were unable to effectively trap C. reinhardtii cells due to the weak acoustic radiation forces generated. Conversely, at high input power levels, the system experiences a rapid temperature increase, posing a risk of cell damage. SAW tweezers, with their localized surface forces, are well suited for precise manipulation at smaller scales, such as in microfluidic systems or on delicate substrates. On the other hand, BAW tweezers are advantageous when manipulation throughout the bulk of the fluid is desired, and they are suitable for larger volumes and higher particle concentrations (20). Here, Cui et al. (5) cleverly combined the advantages of both SAW actuation and BAW trapping array to develop a hybrid BAW/SAW acoustic tweezers platform which can benefit from the precise control of SAW and the larger trapping forces of BAW (Fig. 1). This combined approach allows for effective trapping of swimming cells with both precision and efficiency while minimizing the risk of thermal damage or excessive heating.

Fig. 1.

(A) Schematic of the hybrid BAW/SAW acoustofluidic device from Cui et al. (5). A one-dimensional (1D) standing wave is generated by linearly coupled interdigitated transducers (IDTs) to develop a two-dimensional (2D) bulk acoustic field in a square glass microchamber oriented at 45° to the SAW propagation. In this way, the two overlapping orthogonal standing BAWs (in the X and Y directions) yield an array of trapping sites, similarly to the case of surface wrinkled topographies (21). Particles with a positive acoustic contrast factor, like Chlamydomonas reinhardtii cells, are pushed to these pressure nodes and become trapped. (B) The hybrid BAW/SAW device is capable of imaging and characterizing the swimming dynamics of microswimmers such as C. reinhardtii. Moreover, motility parameters such as ciliary beat frequency and synchrony can be investigated as a function of external factors like temperature and fluid viscosity. The image was created with https://www.biorender.com/.

The described contactless trapping method proved successful in quantifying the motion of cilia and cell bodies in C. reinhardtii cells. The researchers (5) specifically focused on investigating the impact of environmental variables, such as temperature and viscosity, on various aspects of ciliary beating, synchronization, and the three-dimensional helical swimming behavior of C. reinhardtii. Chlamydomonas flagella exhibit a unique “breaststroke” motion, characterized by beat frequencies ranging from 50 to 80 Hz. Additionally, due to inherent asymmetry in the ciliary/flagellar beating pattern, the cell undergoes a gradual rotation (approximately 2 Hz) along its major body axis. Using the acoustofluidic platform, the authors were able to characterize the waveform of both the cis (proximal to the eye spot) and trans cilia in the wild-type biciliate, as well as the single cilium in the uni1 mutant cells. The findings revealed that during asynchronous beating, the waveform of the trans cilium in the wild-type cells closely resembled that of the single cilium in the uniciliated mutant strain. These observations provide insights into the similarities and differences in ciliary waveforms between wild-type biciliate cells and uniciliated mutant cells, thereby gaining a deeper understanding of the role of ciliary structures and their coordination in the motility and function of C. reinhardtii cells. Moreover, the rotational motion of the cell body could be investigated and correlated with an increase in beat frequency resulting from a rise in temperature, suggesting a connection between environmental factors and the dynamics of ciliary motion.

Cui et al. (5) also investigated the impact of mechanical loading on ciliary motion in C. reinhardtii cells, which was obtained by increasing the viscosity of the cell medium. The higher viscosity of the medium resulted in asynchronous beating of the cilia in biciliate cells, suggesting that the increased mechanical load disrupted the coordinated beating pattern of the cilia, causing them to beat independently of each other. Furthermore, the increased viscosity affected the characteristic “corkscrew” motion observed in uniciliate mutants. Additionally, the researchers examined the response of cilia to a transient mechanical stimulus in uniciliate cells. They achieved this by inducing forced translation of the cell through acoustic actuation. The response of the cilia and cell body rotation to this mechanical stimulus was assessed. Interestingly, the forced translation of the cell induced by acoustic actuation had varied effects on cell body rotation. It was observed that the acoustic actuation could either assist or inhibit the rotation of the cell body. By employing this approach, the researchers successfully evaluated the response of swimming behavior and ciliary dynamics to mechanical perturbations, providing insights into how mechanical factors, such as viscosity and transient mechanical stimuli, can influence ciliary motion and the overall swimming characteristics of C. reinhardtii cells.

In summary, the development of single-cell manipulation techniques, such as the hybrid BAW/SAW acoustic tweezers device created by Cui et al. (5), represents a significant breakthrough in the field of microswimmer analysis and offers new opportunities for investigating ciliary/flagellar dynamics in response to external perturbations, including mechanical, chemical, or optical stimuli. The high spatial and temporal resolution of this noninvasive approach allows for precise control and analysis of single cells without inducing detrimental effects that could otherwise impact cellular behavior and gene expression. The versatility of this platform extends beyond the study of algal cells, enabling researchers to gain deeper insights into various biological processes with potential applications across multiple fields of research.