DLpm1 RpK

where n is the viscosity, R is the diameter of the cell outside the pipette, and m is a constant with a value around 9 (Evans and Yeung, 1989).

Inverted Microscope Ray Diagram

Fig. 2-4. Experimental study of cell response to mechanical forces. Cells are deposited on the stage of an inverted microscope equipped with a video camera. The video output is connected to a digitizer mounted on a desk computer. Cells are aspirated into micropipettes connected to a syringe mounted on a syringe holder. Pressure is monitored with a sensor connected to the computer. Pressure and time values are superimposed on live cell images before recording on videotapes for delayed analysis. From Richelme et al., 2000.

Fig. 2-4. Experimental study of cell response to mechanical forces. Cells are deposited on the stage of an inverted microscope equipped with a video camera. The video output is connected to a digitizer mounted on a desk computer. Cells are aspirated into micropipettes connected to a syringe mounted on a syringe holder. Pressure is monitored with a sensor connected to the computer. Pressure and time values are superimposed on live cell images before recording on videotapes for delayed analysis. From Richelme et al., 2000.

Micropipette Aspiration Erythrocyte
Fig. 2-5. Aspiration of a flaccid (a) and swollen (b) red blood cell into a pipette. The diameter of the flaccid cell is approximately 8 |im and that of the swollen cell is about 6 |im. The scale bars indicate 5 |im. From Hochmuth, 2000.
Flaccid Cell Diagram
Fig. 2-6. Diagram for a device for compression of a cell between microplates. Variations of this design also allow for imposition of shear deformation. From Caille et al., 2002.

Shearing and compression between microplates

For cells that normally adhere to surfaces, an elegant but technically challenging method to measure viscoelasticity is by attaching them at both top and bottom to glass surfaces that can be moved with respect to each other in compression, extension, or shear (Thoumine et al., 1999). A schematic diagram of such a system is shown in Fig. 2-6.

In this method a cell such as a fibroblast that adheres tightly to glass surfaces coated with adhesion proteins such as fibronectin is grown on a relatively rigid plate; a second, flexible plate is then placed on the top surface. Piezo-driven motors displace the rigid plate a known distance to determine the strain, and the deflection of the flexible microplate provides a measure of the stress imposed on the cell surface. Use of this device to provide well-defined strains with simultaneous imaging of internal structures such as the nucleus provides a measure of the elastic modulus of fibroblasts around 1000 Pa, consistent with measurements by AFM, and has shown that the stiffness of the nucleus is approximately ten times greater than that of the cytoplasmic protein networks (Caille et al., 2002; Thoumine and Ott, 1997). A recent refinement of the microcantilever apparatus allows a cell in suspension to be captured by both upper and lower plates nearly simultaneously and to measure the forces exerted by the cell as it begins to spread on the glass surfaces (Desprat et al., 2005).

Fluid flow

Cells have to withstand direct mechanical deformations through contact with other cells or the environment, but some cells are also regularly exposed to fluid stresses, such as vascular endothelial cells in the circulating system or certain bone cells (osteocytes) within the bone matrix. Cells sense these stresses and their responses are crucial for many regulatory processes. For example, in vascular endothelial cells, mechanosensing is believed to control the production of protective extracellular matrix (Barbee et al., 1995; Weinbaum et al., 2003); whereas in bone, mechanosensing is at the basis of bone repair and adaptive restructuring processes (Burger and Klein-Nulend, 1999; Wolff, 1986). Osteocytes have been studied in vitro after extraction from the bone matrix in parallel plate flow chambers (Fig. 2-7). Monolayers of osteocytes coated onto one of the chamber surfaces were exposed to shear stress while the

Fluid Flow Device Osteocyte

Fig. 2-7. Fluid flow system to stimulate mechanosensitive bone cells, consisting of a culture chamber containing the cells, a pulse generator controlling the fluid flow, and flow meters. The response of the cells is either biochemically measured from the cells after the application of flow (for example prostaglandin release) or measured in the medium after flowing over the cells (for example nitric oxide). From Klein-Nulend et al., 2003.

Fig. 2-7. Fluid flow system to stimulate mechanosensitive bone cells, consisting of a culture chamber containing the cells, a pulse generator controlling the fluid flow, and flow meters. The response of the cells is either biochemically measured from the cells after the application of flow (for example prostaglandin release) or measured in the medium after flowing over the cells (for example nitric oxide). From Klein-Nulend et al., 2003.

response was measured by detecting the amount of nitric oxide produced as a function of fluid flow rate (Bacabac et al., 2002; Rubin and Lanyon, 1984).

The strain field within individual surface-attached cells in response to shear flow has been mapped in bovine vascular endothelial cells with the help of endogenous fluorescent vimentin (Helmke et al., 2003; Helmke et al., 2001). It was found that the spatial distribution of strain is rather inhomogeneous, and that strain is focused to localized areas within the cells. The method can only measure strain and not stress. The sites for mechanosensing might be those where strain is large if some large distortion of the sensing element is required to create a signal, in other words, if the sensor is "soft." On the other hand, the sites for sensing might also be those where stress is focused and where little strain occurs if the sensing element requires a small distortion, or is "hard," and functions by having a relatively high force threshold.

Numerical simulations can be applied to both the cell and the fluid passing over it. A combination of finite element analysis and computational fluid dynamics has been used to model the flow across the surface of an adhering cell and to calculate the shear stresses in different spots on the cell (Barbee et al., 1995; Charras and Horton, 2002). This analysis provides a distribution of stress given a real (to some resolution) cell shape, but without knowing the material inhomogeneities inside, the material had to be assumed to be linear elastic and isotropic. The method was also applied to model stress and strain distributions inside cells that were manipulated by AFM, magnetic bead pulling or twisting, and substrate stretching, and proved useful to compare the effects of the various ways of mechanical distortion.

Slide

Condenser

Microscope objective, high NA

Fig. 2-8. Schematic diagram of an optical trap.

Quadrant detector

Condenser

Microscope objective, high NA

Fig. 2-8. Schematic diagram of an optical trap.

Optical traps

Optical traps (see Fig. 2-8) use a laser beam focused through a high-numerical aperture microscope objective lens to three-dimensionally trap micron-sized refractile particles, usually silica or latex beads (Ashkin, 1997; Svoboda and Block, 1994). The force acting on the bead at a certain distance from the laser focus is in general very difficult to calculate because (1) a high-NA laser focus is not well approximated by a Gaussian, and (2) a micron-sized refractive particle will substantially affect the light field. Approximations are possible for both small particles (Rayleigh limit) and large particles (ray optics limit) with respect to the laser wave length. For a small particle, the force can be subdivided into a "gradient force" pulling the particle towards the laser focus and a scattering force pushing it along the propagation direction of the laser (Ashkin, 1992). Assuming a Gaussian focus and a particle much smaller than the laser wavelength, the gradient forces in radial and axial direction are (Agayan etal., 2002):

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