Yoichiroh Hosokawa

In this study, we present a novel microfluidic platform for tunable size-based particle separation within a straight microchannel using symmetrical viscoelastic sheath flows. The device incorporates two pairs of symmetrical microchannels for sheath fluid injection: the first pair facilitates particle focusing and separation, while the second pair enables dynamic regulation of the separation distance between particle streams. Experimental results demonstrate that a 50 ppm polyethylene oxide (PEO) solution focuses 1 μm polystyrene particles toward the channel centerline via elastic forces, whereas 5 μm particles migrate toward the channel sidewalls under dominant inertial forces, effectively overcoming the elastic effects. The interplay between inertial and elastic forces thus achieves size-dependent particle separation. Furthermore, by adjusting the flow rate of the PEO sheath in the second pair of microchannels, the separation distance between the two particle populations can be modulated in real time. Higher PEO concentrations (500 and 1000 ppm) exhibit enhanced capabilities to deflect particle flow streams. By contrast, the lower PEO concentrations like 50, 100 and 200 ppm are more versatile in adjusting the separation distance. The biological applicability of this platform is further demonstrated through the tunable separation of Escherichia coli (E. coli) and Chlorella vulgaris (C. vulgaris). This microfluidic device demonstrates significant potential for downstream particle processing applications, including real-time particle detection and targeted drug delivery.

Main conclusion Our method can simultaneously quantify cell wall elasticity and turgor pressure of live plant cells through AFM measurements and calculations based on elastic shell theory. Abstract The morphological behavior of plant cells depends on their mechanical properties. Cell wall elasticity (E) and turgor pressure (P) are main factors that dominate the behavior. A method to simultaneously quantify them in live cells has yet to be established, hindering progress in plant mechanobiology. Recently, atomic force microscopy (AFM) has been used to analyze single cells based on Hertz’s contact theory (HCT). However, HCT cannot evaluate P. Several groups have attempted to evaluate P by adapting elastic shell theory (EST), but it is still difficult to estimate both E and P from the indentation data and EST alone. Herein an analytical method is proposed based on EST using the cell indentation and surface geometry from the AFM measurements. We also demonstrate the reliability of our approach under various osmotic pressure conditions and simultaneously determine the values of P and E in epidermal monolayer cells of an Allium cepa L.

Impedance cytometry is wildly used in single-cell detection, and its sensitivity is essential for determining the status of single cells. In this work, we focus on the effect of electrode gap on detection sensitivity. Through comparing the electrode span of 1 µm and 5 µm, our work shows that narrowing the electrode span could greatly improve detection sensitivity. The mechanism underlying the sensitivity improvement was analyzed via numerical simulation. The small electrode gap (1 µm) allows the electric field to concentrate near the detection area, resulting in a high sensitivity for tiny particles. This finding is also verified with the mixture suspension of 1 µm and 3 µm polystyrene beads. As a result, the electrodes with 1 µm gap can detect more 1 µm beads in the suspension than electrodes with 5 µm gap. Additionally, for single yeast cells analysis, it is found that impedance cytometry with 1 µm electrodes gap can easily distinguish budding yeast cells, which cannot be realized by the impedance cytometry with 5 µm electrodes gap. All experimental results support that narrowing the electrode gap is necessary for tiny particle detection, which is an important step in the development of submicron and nanoscale impedance cytometry.

The stiffness of a plant cell in response to an applied force is determined not only by the elasticity of the cell wall but also by turgor pressure and cell geometry, which affect the tension of the cell wall. Although stiffness has been investigated using atomic force microscopy (AFM) and Young’s modulus of the cell wall has occasionally been estimated using the contact-stress theory (Hertz theory), the existence of tension has made the study of stiffness more complex. Elastic shell theory has been proposed as an alternative method; however, the estimation of elasticity remains ambiguous. Here, we used finite element method simulations to verify the formula of the elastic shell theory for onion (Allium cepa) cells. We applied the formula and simulations to successfully quantify the turgor pressure and elasticity of a cell in the plane direction using the cell curvature and apparent stiffness measured by AFM. We conclude that tension resulting from turgor pressure regulates cell stiffness, which can be modified by a slight adjustment of turgor pressure in the order of 0.1 MPa. This theoretical analysis reveals a path for understanding forces inherent in plant cells.

Axon outgrowth is promoted by the mechanical coupling between the dynamic actin cytoskeleton and adhesive substrates via clutch and adhesion molecules in the axonal growth cone. In this study, we utilized a femtosecond laser-induced impulse to break the coupling between an axonal growth cone and an adhesive substrate, enabling us to evaluate the strength of the binding between proteins in the growth cone and a laminin substrate, and also determine the contribution of adhesion strength to axon outgrowth. We found that the adhesion strength of axonal L1 cell adhesion molecule (L1CAM)-laminin binding increased with the density of the laminin substrate. In addition, fluorescent speckle microscopy revealed that the retrograde flow of actin filaments in the axonal growth cone was dependent on the laminin density such that the flow speed reduced with increasing L1CAM-laminin binding. However, axon outgrowth did not increase monotonically with increased L1CAM-laminin binding but rather exhibited biphasic behavior, in which the outgrowth was suppressed by excessive L1CAM-laminin binding. Our quantitative evaluations of the adhesion strength suggest that the biphasic outgrowth is regulated by the balance between traction force and adhesion strength as a result of changes in the number of L1CAM-laminin interactions. These results imply that adhesion modulation is key to the regulation of axon guidance. Significance Statement There is a lack of a method to evaluate an adhesion strength of axonal growth cone. We evaluated the adhesion strength of axonal growth cones to a substrate by utilizing the force applied from a femtosecond laser impulse. This study shows that the adhesion strength between the growth cone and substrate is strengthened by L1CAM-laminin binding. Axon outgrowth did not increase monotonically with increased L1CAM-laminin binding but rather exhibited biphasic behavior, indicating that existence of suitable adhesion strength for axonal growth. Our findings suggest that the balance between growth cone adhesion strength and the traction force transmitted via cytoskeletal flow is a key factor in axon guidance.

Continuous microfluidic focusing of particles, both synthetic and biological, is significant for a wide range of applications in industry, biology and biomedicine. In this study, we demonstrate the focusing of particles in a microchannel embedded with glass grooves engraved by femtosecond pulse (fs) laser. Results showed that the laser-engraved microstructures were capable of directing polystyrene particles and mouse myoblast cells (C2C12) towards the center of the microchannel at low Reynolds numbers (Re < 1). Numerical simulation revealed that localized side-to-center secondary flows induced by grooves at the channel bottom play an essential role in particle lateral displacement. Additionally, the focusing performance proved to be dependent on the angle of grooves and the middle open space between the grooves based on both experiments and simulation. Particle sedimentation rate was found to critically influence the focusing of particles of different sizes. Taking advantage of the size-dependent particle lateral displacement, selective focusing of micrometer particles was demonstrated. This study systematically investigated continuous particle focusing in a groove-embedded microchannel. We expect that this device will be used for further applications, such as cell sensing and nanoparticle separation in biological and biomedical areas.

Mechanical properties of cells and tissues closely link to their architectures and physiological functions. To obtain the mechanical information of submillimeter scale small biological objects, we recently focused on the object vibration responses when excited by a femtosecond laser-induced impulsive force. These responses are monitored by the motion of an AFM cantilever placed on top of a sample. In this paper, we examined the surface cellular stiffness of zebrafish embryos based on excited vibration forms in different cytoskeletal states. The vibration responses were more sensitive to their surface cellular stiffness in comparison to the Young’s modulus obtained by a conventional AFM force curve measurement.