Investigating mechanical properties at the tissue, cellular, and subcellular levels is crucial in microbiology, cell biology, developmental biology, and medicine. Numerous methods exist, including magnetic twisting cytometry, particle-tracking microrheology, optical stretching, parallel-plate rheology, and indentation techniques like atomic force microscopy (AFM) and cellular force microscopy (CFM). However, these methods often lack robust manipulation capabilities, limiting their ability to access all regions of a biological sample. 3D mechanical quantification is particularly restricted at the microscale, necessitating assumptions and simplifications in modeling complex systems. Ensemble averaging from multiple individuals to achieve complete specimen coverage also reduces experimental accuracy. While various techniques using magnetism, hydrodynamics, or optical fields can rotate microscale objects, many rely on specific specimen properties, limiting their biological applicability. Acoustic waves, known for their biocompatibility and controllability, offer an alternative. This research combines micro-indentation with an acoustically driven, bubble-based device for non-invasive trapping and 3D characterization of micron-sized objects. The integration of 3D acoustic manipulation with micro-indentation allows for the quantification of different surface regions of single specimens, expanding the possibilities of biological research at the microscale. The study first describes the system's structure and operation, quantifying manipulation capabilities. It then demonstrates the advantages of this method by mechanically characterizing plant cell walls and subsequently expands the application to the organism level, investigating the influence of internal organs on the mechanical properties of surrounding tissue in nematodes.

The paper reviews existing methods for characterizing cell mechanics, highlighting their limitations in achieving 3D characterization of individual specimens. It discusses existing methods that utilize magnetism, hydrodynamics, or optical fields for microscale object rotation, noting their limitations in applicability to diverse biological samples. The authors highlight the advantages of using acoustic waves for manipulation due to their biocompatibility and controllability, citing previous research on acoustofluidic systems for particle and sample manipulation and rotation. The literature review sets the stage for the proposed method by emphasizing the need for a technique that combines robust manipulation with precise mechanical characterization at the microscale.

The researchers developed a system integrating an acoustically driven, bubble-based manipulation device with a cellular force microscopy (CFM) setup. The acoustic manipulation device uses an open-microchannel arrangement with linear arrays of rectangular micro-cavities to trap and rotate samples using acoustically actuated microbubbles. The microbubbles generate microstreaming, which rotates the specimen, allowing the force probe to access different regions of the sample. The CFM system measures the applied force and displacement during micro-indentation, calculating the apparent stiffness. The system uses a commercially available MEMS-based capacitive force sensor and a 3D positioner to precisely control the force probe's position. The study used *Lilium longiflorum* pollen grains and *C. elegans* nematodes as model systems. For pollen grains, the researchers measured apparent stiffness at various surface regions (exine and intine) in deionized water and a calcium chloride solution, comparing results to dehydrated specimens. Finite element method (FEM)-based simulations were used to analyze the complex interplay of geometry, heterogeneous material composition, and turgor pressure on apparent stiffness. For *C. elegans*, the researchers used acoustic manipulation to rotate the nematodes, performing micro-indentations along their longitudinal axis to assess variations in apparent stiffness. The experimental setup is detailed, including the fabrication process of the acoustic manipulation device, sample preparation, and data acquisition and analysis methods, along with statistical methods used to evaluate the results. The FEM simulations were performed using MorphoMechanX, modeling the pollen grain as a pressurized multi-layered structure with different material properties assigned to the intine and exine layers.

The study successfully demonstrated the 3D mechanical characterization of single cells and small organisms using the combined acoustic manipulation and CFM system. For *Lilium longiflorum* pollen grains, the researchers found significant differences in apparent stiffness between the intine (softer) and exine (stiffer) regions. The average apparent stiffness of the intine was 9.3 ± 4.4 N/m, and the exine was 16.5 ± 6.6 N/m. The ratio of intine to exine stiffness (k_i/k_e) was 0.56 ± 0.1 in deionized water. Addition of CaCl₂ significantly increased this ratio (p=0.000312), suggesting Ca²⁺-mediated stiffening of the intine. Dehydrated pollen grains exhibited significantly higher stiffness (194 N/m) than hydrated grains, indicating hydration-induced changes in the cell wall matrix. FEM simulations helped disentangle the contributions of turgor pressure and material properties to the measured apparent stiffness, estimating a material stiffness ratio (E_e/E_i) of approximately 10. For *C. elegans*, 3D characterization revealed large variations in apparent stiffness along the nematode's body (0.53 ± 0.07 N/m for softer regions and 0.75 ± 0.11 N/m for stiffer regions, p=3.14e-10), potentially reflecting the influence of internal organs like body wall muscles. This highlights the importance of 3D characterization to avoid misinterpretations from 1D measurements.

The findings address the need for a robust method for 3D mechanical characterization of microscale biological samples. The combined acoustic manipulation and CFM system provides a versatile tool for studying the mechanical properties of single cells and small organisms, overcoming the limitations of existing techniques. The results highlight the importance of considering local variations in mechanical properties and the impact of environmental factors (hydration, Ca²⁺ concentration) on cell wall stiffness in pollen grains. The observed variations in apparent stiffness in *C. elegans* underscore the necessity of 3D analysis for accurate assessment of tissue mechanics in multicellular organisms. The FEM simulations provide valuable insights into the complex relationship between apparent stiffness and underlying material properties and turgor pressure. This work opens up new avenues for biophysical modeling and understanding the biomechanics of living systems at the microscale.

This study presents a novel platform for 3D mechanical characterization of microscale biological samples, combining acoustic manipulation with cellular force microscopy. The method successfully characterized the mechanical properties of *Lilium longiflorum* pollen grains and *C. elegans* nematodes, revealing previously inaccessible information about local variations in stiffness and the influence of environmental factors. The results highlight the importance of 3D analysis for accurate biomechanical modeling. Future research could explore the application of this technique to other biological systems and further refine the biophysical models to better understand the complex interplay of material properties and cellular processes.

The temporal stability of the microbubbles is limited, leading to variations in rotational velocity over time. The FEM simulations involved simplifications in representing the pollen grain structure, potentially affecting the accuracy of material property estimations. In *C. elegans* studies, the use of a paralyzing drug may have influenced the measured stiffness values, warranting further investigation without drug application. While the study shows high repeatability in measurements (average coefficient of variation of 4.8%), further investigations on the bimodal behavior observed in some samples are needed.