Among the methods for the determination of mechanical properties of living cells acoustic microscopy provides some extraordinary advantages. It is relatively fast, of excellent spatial resolution and of minimal invasiveness. Sound velocity is a measure of the stiffness or Young's modulus of the cell. Attenuation of cytoplasm is a measure of supramolecular interactions. These parameters are of crucial interest for studies of cell motility, volume regulations and to establish the functional role of the various elements of the cytoskeleton. Using a phase and amplitude sensitive modulation of a scanning acoustic microscope (Hillman et al., 1994, J. Alloys Compounds. 211/212:625-627) longitudinal wave speed, attenuation and thickness profile of a biological cell are obtained from the voltage versus frequency or V(f) curves. A series of pictures, for instance in the frequency range 980-1100 MHz with an increment of 20 MHz, allows the experimental generation of V(f) curves for each pixel while keeping the lens-specimen distance unchanged. Both amplitude and phase values of the V(f) curves are used for obtaining the cell properties and the cell thickness profile. The theoretical analysis shows that the thin liquid layer, between the cell and the substrate, has a strong influence on the reflection coefficient and should not be ignored during the analysis. Cell properties, cell profile and the thickness of the thin liquid layer are obtained from the V(f) curves by the simplex inversion algorithm. The main advantages of this new method are that imaging can be done near the focal plane, therefore an optimal signal to noise ratio is achieved, no interference with Rayleigh waves occurs, and the method requires only an approximate estimate of the material properties of the solid substratum where the cells are growing on.

The shape of cells during interphase in sparse cultures often resembles that of fried eggs. XTH-2 cells, which have been derived from tadpole heart endothelia, provide a typical example of this type of shape. To understand the physical basis of this shape, the cytoskeleton of these cells has been investigated in detail. Subcellular elasticity data have been achieved by scanning acoustic microscopy (SAM). Their changes were observed during treatment of the cells with microtubule-disrupting agents (colcemid and low temperature), and shape generation in giant cells produced by electro-fusion was observed with SAM, revealing the role of the nucleus as a force centering organelle. From these observations combined with well-documented observations on cellular dynamics described in the literature, a model is developed explaining the fried-egg shape of cells by means of interacting forces and fluxes (cortical flow, bulk flow of cytoplasm, microtubule-mediated transport of cytoplasm) of cytoplasm. The model also allows the comprehension of the increase of tension in cells treated with colcemid.

The state of crosslinking of microfilaments and the state of myosin-driven contraction are the main determinants of the mechanical properties of the cell cortex underneath the membrane, which is significant for the mechanism of shaping cells. Therefore, any change in the contractile state of the actomyosin network would alter the mechanical properties and finally result in shape changes. The relationship of microtubules to the mechanical properties of cells is still obscure. The main problem arises because disruption of microtubules enhances acto-myosin-driven contraction. This reaction and its impact on cell shape and elasticity have been investigated in single XTH-2 cells. Microtubule disruption was induced by colcemid, a polymerization inhibitor. The reaction was biphasic: a change in cell shape from a fried egg shape to a convex surface topography was accompanied by an increase in elastic stiffness of the cytoplasm, measured as longitudinal sound velocity revealed by scanning acoustic microscope. Elasticity increases in the cell periphery and reaches its peak after 30 min. Subsequently while the cytoplasm retracts from the periphery, longitudinal sound velocity (elasticity) decreases. Simultaneously, a two- to threefold increase of F-actin and alignment of stress fibers from the cell center to cell-cell junctions in dense cultures are induced, supposedly a consequence of the increased tension.

The subtraction of subsequent scanning acoustic microscope images (SubSAM) of living cells taken in distinct time intervals reveals subcellular motility domains that are dependent on metabolic energy and correspond to cell surface deformations like protrusions, ruffling, and microblebs. This motility can be quantitated by determining the changes of the grey levels vs. time. Tension has been postulated as a global parameter in the control of cell shape and cell surface motility [Albrecht‐Bühler 1987: Cell Motil. Cytoskeleton 7:54–67; Bereiter‐Hahn et al., 1995: Biochem. Cell Biol. 73:337–348; Sheetz and Dai, 1996: Trends Cell Biol. 6:85–89]. For direct evaluation, the activity of the motility domains was measured while applying external tension (stretching) or internal tension (contraction induced by nocodazole) and by relaxation due to desintegration of the actin‐cytoskeleton using low concentrations of cytochalasin D (0.5 μg/ml). Elevated tension, regardless of how it is generated, externally or internally, whether directed or isotropic, lowers cell surface motility. In contrast, the relaxation of the cell cortex by cytochalasin D increases cell surface motility. Thus, a direct relationship between tension at the cell surface and surface motility was established as has been suggested by Sheetz and Dai [1996: Trends Cell Biol. 6:85–89] Cell Motil Cytoskeleton 43:349–359, 1999. © 1999 Wiley‐Liss, Inc.

This paper reports preliminary results of the observation by acoustic microscopy of living cells in vitro. The scanning acoustic microscope uses high-frequency sound waves to produce images with submicrometer resolution. The contrast observed in acoustic micrographs of living cells depends on the acoustic properties (i.e., density, stiffness, and attenuation) and on the topographic contour of the cell. Variation in distance separating the acoustic lens and the viewed cell also has a profound effect on the image. When the substratum is located at the focal plane, thick regions of the cell show a darkening that can be related to cellular acoustic attenuation (a function of cytoplasmic viscosity). When the top of the cell is placed near the focal plane, concentric bright and dark rings appear in the image. The location of the rings can be related to cell topography, and the ring contrast can be correlated to the stiffness and density of the cell. In addition , the character of the images of single cells varies dramatically when the substratum upon which they are grown is changed to a different material. By careful selection of the substratum, the information content of the acoustic images can be increased. Our analysis of acoustic images of actively motile cells indicates that leading lamella are less dense or stiff than the quiescent trailing processes of the cells. The scanning acoustic reflection microscope is a novel way of investigating biological materials with submicrometer resolution. The information content of acoustic images differs from that of optical or electron microscopic images. Acoustical images contain information about mechanical properties of the object: density, stiffness, and acoustic attenuation. An acoustic microscope with water as the coupling medium has been used to image subcellular detail in fixed cells (1). A liquid argon-coupled acoustic microscope has been used to image human meta-phase chromosomes with a resolution of 0.38 ttm (2). In this paper we present preliminary results of the observation of living cells in vitro. We have found that living cells can be viewed for several hours with no apparent disruption. The cells continue to exhibit normal patterns of motility and remain well spread on the substratum. It appears that neither the high-frequency sound waves nor the mechanical scanning cause significant cellular damage. Living cells are particularly interesting for study because little is known about the mechanical properties of a single cell and their relations to cell behavior. The acoustic microscope used in this study was developed by Lemons and Quate (3) and was modified to operate as a reflection instrument at nearly opitcal wavelengths by Jipson and Quate (4). The basic operation of a reflection acoustic microscope can be understood with reference to Fig. 1. A radio-frequency electrical pulse is used to excite a thin-film piezoelectric transducer located at one end of a sapphire crystal. The transducer generates an acoustic plane wave which propagates through the sapphire...