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The mechanical properties of cells reflect dynamic changes of cellular organization which occur during physiologic activities like cell movement, cell volume regulation or cell division. Thus the study of cell mechanical properties can yield important information for understanding these physiologic activities. Endothelial cells form the thin inner lining of blood vessels in the cardiovascular system and are thus exposed to shear stress as well as tensile stress caused by the pulsatile blood flow. Endothelial dysfunction might occur due to reduced resistance to mechanical stress and is an initial step in the development of cardiovascular disease like, e.g., atherosclerosis. Therefore we investigated the mechanical properties of primary human endothelial cells (HUVEC) of different age using scanning acoustic microscopy at 1.2 GHz. The HUVECs are classified as young (tD< 90 h) and old (tD> 90 h) cells depending upon the generation time for the population doubling of the culture (tD). Longitudinal sound velocity and geometrical properties of cells (thickness) were determined using the material signature curve V(z) method for variable culture condition along spatial coordinates. The plane wave technique with normal incidence is assumed to solve two-dimensional wave equation. The size of the cells is modeled using multilayered (solid-fluid) system. The propagation of transversal wave and surface acoustic wave are neglected in soft matter analysis. The biomechanical properties of HUVEC cells are quantified in an age dependent manner.

A new technique for investigating the surface plasmons by means of a scanning acoustic microscope is proposed. Within this technique, the surface electromagnetic wave (plasmon polariton) is excited by laser radiation on one side of a metal film, while a scanning acoustic microscope excites surface acoustic waves on the other side of the film. Obtained for the first time, the acoustic images of plasmons, propagating on the grating surface, demonstrate the possibility of studying the plasmon wave field distribution by means of a scanning acoustic microscope.

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 980–1100MHz with an increment of 20MHz, 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.

A nondestructive method to determine viscoelastic properties of gels and fluids involves an oscillating glass fiber serving as a sensor for the viscosity of the surrounding fluid. Extremely small displacements (typically 1–100nm) are caused by the glass rod oscillating at its resonance frequency. These displacements are analyzed using a phase-sensitive acoustic microscope. Alterations of the elastic modulus of a fluid or gel change the propagation speed of a longitudinal acoustic wave. The system allows to study quantities as small as 10μl with temporal resolution >1Hz. For 2–100μM f-actin gels a final viscosity of 1.3–9.4mPa s and a final elastic modulus of 2.229–2.254GPa (corresponding to 1493–1501m/s sound velocity) have been determined. For 10- to 100-μM microtubule gels (native, without stabilization by taxol), a final viscosity of 1.5–124mPa s and a final elastic modulus of 2.288–2.547GPa (≅1513–1596m/s) have been determined. During polymerization the sound velocity in low-concentration actin solutions increased up to +1.3m/s (≅1.69kPa) and decreased up to −7m/s (≅49kPa) at high actin concentrations. On polymerization of tubulin a concentration-dependent decrease of sound velocity was observed, too (+48 to −12m/s ≅ 2.3–0.1MPa, for 10- to 100-μM tubulin). This decrease was interpreted by a nematic phase transition of the actin filaments and microtubules with increasing concentration. 2mM ATP (when compared to 0.2mM ATP) increased polymerization rate, final viscosity and elastic modulus of f-actin (17μM). The actin-binding glycolytic enzyme hexokinase also accelerated the polymerization rate and final viscosity but elastic modulus (2.26GPa) was less than for f-actin polymerized in presence of 0.2mM ATP (2.28GPa).

A method is described for the determination of cellular F-actin content using fluorometry of TRITC-phalloidin at very high dilution. In this case no saturation of all binding sites available is reached, however the staining is highly specific and the specificity is not affected by the preparative procedure as may be the case at high concentrations of TRITC-phalloidin. The method is based on calculation of fluorescence intensity at equilibrium conditions from measurements at two different numbers of exchange of the staining solution by using Lineweaver-Burk plotting.

The scanning of fluorescence of a cell culture along a path several milimeters long, gives a series of signals, which allow calculation of the fluorescence emitted per cell. The emission at 450±10 nm, excited at 360 nm, provides a measure of NADH (and NADPH) per cell. Combination of this method with modifications in energy metabolism, allows determination in the same sample of total NAD content, of NAD redox state, content of NAD in the mitochondria and content of NAD plus NADP in the cytoplasm. The method can be extended to measure other cellular constituents by labelling with fluorescent markers, e.g. antibodies.

A population of endothelial cells growth from Xenopus laevis tadpole hearts was investigated by microinterferometry. The resulting interferograms were evaluated by an automatic image analyzer. The mean values of dry mass were 778±340 pg (10-15 g) for whole cells, 648±309 pg for cytoplasm, 116±45 pg for nuclei, and 19±10 pg for nucleoli. Two subpopulations of cells were identified, an actively growing one and a less active one. The density (dry mass per µm2) of the nuclei and nucleoli of less active cells was greater than that of the nuclei and nucleoli of actively growing cells. In addition, the inactive cells were always large and possessed a considerable amount of cytoplasm. The entrance of cells into S-phase could not be detected by microinterferometry; and no differences were apparent between cells possessing one nucleolus and those containing two nucleoli. The values obtained in these amphibian cells were compared with those derived from mammalian cells.

FITC-Con A-fluorescence and electron microscopy have been used in parallel for the visualization of rough endoplasmic reticulum inXenopus laevis tadpole heart primary tissue culture cells. rER distribution pattern as revealed by both methods is compared by superimposition of images of the same cell. Coincidence of rER distribution is established.

In vivo, epidermal cells are committed to terminal differentiation in which they undergo a series of morphological and biochemical changes. In vitro, keratinocytes are able to undergo some steps of this differentiation process only. In view of the fact that in vivo skin is continuously subjected to mechanical stress, we investigated the stimulation of differentiation of transformed keratinocytes by mechanical stimulation. The cells, grown in plastic culture dishes, were periodically treated with weights exerting a pressure of 0.015 Ncm-2. This stimulation lasted from 1 to 4 days. Then keratinocytes were examined using indirect immunofluorescence, 3H-thymidine and 14C-amino acid incorporation, SDS polyacrylamide gel electrophoresis, and Western blotting. Following pressure treatment, the previously monolayered keratinocytes locally grew up to several layers, the number of horny scales increased and, after 4 days, the pattern of cytokeratin was modified. The total amount of keratin increased, forming granular accumulations, while the proliferation rate of the cells decreased. Both the 67 kDa and 49.5 kDa keratin subunits increased in stimulated cells. Moreover, a weak keratin band of 44 kDa appeared that was not present in controls. The results demonstrate that cyclic pressure promotes differentiation of cultivated epidermal cells.

Immunofluorescence and electron microscopy were used to analyze the relationships between the organization of collagen fibrils in elasmoid scales, and the orientation of microtubules and actin microfilaments in the scleroblasts producing this collagenous stroma. Attention was focused on the basal plate of the scales because of the highly ordered three-dimensional arrangement of the collagen fibrils in superimposed plies forming an acellular plywood-like structure. The collagen fibrils are synthesized by the scleroblasts forming a monolayered pseudo-epithelium, the hyposquama, at the lowest surface of the scale. Fully developed scales with a low collagen deposition rate were compared with regenerating scales active in fibrillogenesis. When an ordered array of the collagen fibrils is found, the innermost collagen fibrils are coaligned with microtubules and actin microfilaments. Thus, because of this coalignment, microtubules and actin microfilaments of the hyposquamal scleroblasts are subjected to consecutive alterations during the formation of the plies of the basal plate. The sequence of events when the collagen fibrils change their direction from one ply to the other in the basal plate is deduced from immunofluorescence and phase-contrast-microscopic observations. During the formation of the orthogonal plywood-like structure in the regenerating scales, first microtubules may change their curse with a rotating angle of about 90°; then, actin microfilaments are disorganized and reorganized by interacting mechanically with the microtubules with which they are coaligned. Collagen fibrils are synthesized in a direction that is roughly perpendicular to that of the preceding ply. The unknown signals inducing the change in direction of the cytoskeleton may be transmitted throughout the hyposquama via gap junctions.

Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.

In this paper, a mathematical formulation is presented to compute the V(z) of a tapering layered solid and applying this formulation to the determination of acoustic properties of biological cells and tissues. The formulation is adopted in the simplex inversion algorithm to obtain the acoustic properties of a tapering cell from its V(z) values. The influence of two parameters had been considered: The tapering angle and the presence of a thin liquid layer present between cells and the substratum to which they adhere. Up to a tapering angle less than 10°, it can be safely neglected. However, if a larger angle is neglected, then the acoustic wave velocity in the cell is overestimated. Cell thickness estimation is not affected significantly when the tapering angle is ignored. The calculations of acoustic properties of cells are considerably influenced by the introduction of a thin fluid layer between the solid substratum and the overlying cell, neglecting the presence of at least a very thin layer (20–30 nm), in general, results in a considerable overestimation of sound velocity. The reliability of the data calculated from V(z) values was ascertained using an independent method to determine cell thickness by calculating it from the interference fringe pattern obtained with the reflection‐interference light microscope. The shape of the glutaraldehyde‐fixed cells was similar to fried eggs. The highest sound velocities were found close to the periphery of the dome‐shaped cell center. In the very center and over most of the area of the thin periphery, sound velocity was close to that in saline.

Summary Human polymorphonuclear leucocytes and epitheliocytes isolated from tadpole tails ofXenopus laevis were used to observe the responses of cells to mechanical stimulation with a microneedle. Biphasic responses were observed: a retraction phase lasting 1–3 s was followed by an extension phase lasting 10–40s. Weak stimulation evoked alocal response whilst on strong stimulation the whole cells rounded up. Spreading after induced rounding was at least one order of magnitude faster (it lasted less than 1–2min) than cell spreading after chemical dissociation of cell cultures. Local or extended loss of cell attachment to the substratum (observed with reflection interference contrast microscopy) preceded changes in cell morphology, visible with phase contrast microscopy. Repeated weak stimulation of one cell side induced extension and locomotion of the cell in this direction. The reported biphasic responses of cells to mechanical stimulation highlight the significance of exact timing when following any cell response to external stimuli.

Osmotic differences between the cytoplasm and the surrounding fluid belong to the most important factors in shaping an animal tissue cell. This implies an elastic and contractile fibrillar system attached to the cell membrane which counteracts ionic imbalances and determines cell morphology. Therefore cell volume and cell shape are functionally coupled. Many cell types, epithelial cells in particular, are able to control their volume: ie, swelling (or shrinking) induced by varying the osmolarity of the surrounding fluid is compensated and the previous volume is achieved during a time interval of 10-40 min. This compensation reaction includes the opening of ion channels, activation of the Na/K/2C1 co-transport mechanism, and activities of the cytoskeleton. The changes in ion fluxes have been investigated for various cell types. The main question which is still under debate refers to the signal involved in telling a cell to have reached its appropriate volume. One of the most probable factors is the tension in the cell membrane. By action of tension-sensitive ion channels (K+, Ca++-channels) ion fluxes could be modified. Mechanical forces acting on the cell membrane always affect the cortical cytoplasm - membrane complex: In many cell types a fibrillar meshwork is closely connected to the membrane, thus determining its mechanical properties. Destruction of this complex by drugs like cytochalasins alters the mechanics of the cortex and destroys the ability of the cells for volume regulation. If mechanical forces in the cortex are involved in volume control, the loss of this ability would be a reasonable consequence of cytochalasin treatment. Measurement of the forces at the surface of cells during swelling and during the compensation phase is a direct approach to the problem of volume regulation. Scanning acoustic microscopy (SAM) is an elegant method to determine elastic properties on a subcellular level. Measuring the reflection of sound at the surface of a cell gives a good indication of local elasticity (and thus “tension at the surface”). Using the cell line HaCat which was derived from human epidermis, elasticity changes have been followed by SAM during hypotonicity-induced swelling and the subsequent compensatory volume decrease. On the basis of the present results the regulatory volume decrease (RVD) may be achieved by an increase of forces in the cortex by actomyosin-based contractions in addition to altered ion fluxes. Volume increase first results in a destruction of the actin fibrillar system which is immediately restored by stimulated actin polymerisation which strengthens the complex of the plasma membran with the cortical fibrillar layer. The results support the view that tension in the cortex may participate in RVD and be a signal for volume regulation.

A pressure device has been used to obtain information about the forces involved in the maintenance of the aggregated state of melanophores of the angelfish, Pterophyllum scalare. Single aggregated melanophores of isolated scales were submitted to mechanical compression with forces ranging from 50–320 µp. As a function of the gradually increasing force melanophores disperse their pigment, the degree of dispersion being proportional to the intensity of the force. When microtubules are destroyed by treatment with 0.3 mM vinblastine in KCl solution, pigment dispersion in response to the external force is similar to that observed in KCl alone. After changing the medium to NaCl solution, melanin granules remain concentrated in the cell center; the force-induced melanosome dispersion, however, is significantly enhanced. Distinctly lower forces are required to produce an expansion equivalent to that observed in KCl solution. When the medium is changed to vinblastine-KCl again, the dispersion in response to the external force resembles that obtained before NaCl treatment. Removal of Ca++ and Mg++ ions by treatment with 2 mM EDTA orEGTA in Ringer's solution containing 0.1 mM adrenalin produces a remarkable enhancement of melanosome dispersion in response to increasing external force. This effect of EDTA or EGTA is completely reversible. When the medium is changed to normal Ca++-Ringer's, the force/dispersion curve resembles that obtained before EDTA or EGTA treatment.

Cyclic adenosine monophosphate (cAMP) has been shown to cause pigment dispersion in amphibian and fish melanophores. Since pigment displacements in melanophores of Pterophyllum scalare are known to be accompanied by assembly and disassembly of microtubules, the effect of cAMP on this process was investigated. Melanophores of isolated scales were treated with cAMP in the presence of vinblastine, a potent antimicrotubular agent. During the initial phase of vinblastine action, cAMP as well as its dibutyryl derivative are capable of counteracting the inhibitory effects of vinblastine on pigment dispersion. In addition, cAMP retains the velocity of pigment dispersion at about the maximum level during 1 hour experiments. Pigment aggregation was unaffected by cAMP. Since pigment dispersion in Pterophyllum-melanophores is accompanied by assembly of microtubules, it is concluded that cAMP influences, at least in part, melanosome dispersion through facilitation of microtubule assembly.

The effects of the calcium channel blockers S- and R-verapamil or Ro 40-5967 on penetration processes of peripheral blood lymphocytes (PBL) added to allogenic human vascular endothelial cell (HUVEC) monolayers were studied. Both PBL adhesion and migration were evaluated by combined phase contrast and reflection interference contrast microscopy. PBL adhesion was inhibited by 22% when 100 μg/ml verapamil was added to the cell cultures. PBL migration was completely suppressed with verapamil concentrations above 80 μg/ml (EC50: S-verapamil: 59.7 μg/ml, R-verapamil: 52.3 μg/ml). No differences were seen in the results obtained either with or without cytokine (γ-IFN or IL-1) stimulation. Ro 40-5967 redlfced adhesion completely above concentrations of 40 μg/ml. EC50 for migration was 13 ± 2 or 13 ± 1 μg/ml, respectively. Immunohistochemical analysis of adhesion molecule expression on HUVEC revealed no inhibition of 1CAM-1 and VCAM-1 by verapamil or Ro 40-5967. We concluded that the effects of the calcium channel blockers did not depend on cytokine stimulation and, as they were found similar for both verapamil enantiomers, also did not depend on the calcium channel blocking properties of the compounds. Since adhesion molecule expression was not reduced on HUVEC the Ca2+-channel blockers tested in this assay seem to affect cellular infiltration via other pathways.

In density-inhibited 3T3 fibroblast cultures F-actin is localized almost exclusively at the cell-to-cell borders. Depolymerization of microtubules results in a rearrangement of the F-actin pattern. Numerous actin bundles are formed, most of them are anchored to the cell borders. This rearrangement is preceded by a transient rise in F-actin content. Copyright 1993, 1999 Academic Press

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.

The characterization of the fibrillar collagens and the cellular control of their spatial deposition were studied in fish scales using immunofluorescence, electron microscopy, electrophoretic and HPLC analyses, immunoprecipitation and hybridization with cDNA probes. This study was carried out on undisturbed and regenerating scales in situ and in organ and cell cultures from regenerating scales. The hyposquamal scleroblasts forming a pseudoepithelium show an apico-basal polarization and synthesize thick collagen fibrils (100 nm) organized in a plywood pattern as long as the integrity of the cell-cell and cell-collagenous matrix contacts are preserved. In culture, scleroblasts become fibroblast-like and produce an unordered meshwork of thin collagen fibrils (30 nm). Comparison of the synthesized collagens in culture with those extracted from the scales indicates that culture conditions modify fibrillogenesis but do not change the expression of fibrillar collagen genes. Type I collagen, the prédominent component, is associated with the minor type V collagen. Type HI collagen was not present. In type I collagen, a third chain, α3 chain, was identified. The ratio between the 3 chains suggests the coexistence of two heterotrimers (αl(I))2 α2(1) and αl(I) α2(1) α3(I). Analysis by HPLC and electrophoresis of the cyanogen bromide-derived peptides obtained from the purified α3 chain support the hypothesis that αl(I) and α3(I) chains are encoded by two different genes. The presence of the two types of heterotrimers in vivo as well as in vitro could correspond to an innate property of the goldfish scleroblasts. Despite the fact that teleost cyanogen bromide-derived peptides differ from those of higher vertebrates, homologies with the mammalian collagen genes (human, for example) are sufficient to allow the detection of mRNA transcripts for al(I), α2(I) and α2(V) from confluent scleroblast cultures with human probes.

Summary FITC-Con A fluorescence was used to visualize rER arrangement of endothelial cells derived fromXenopus laevis tadpole hearts. In particular determinants of rER organization, intracellular localization and the interrelationships with other organelles were analysed. rER occurs in association with nucleus, mitochondria and microtubules.

Mitochondrial movements have been followed by phase-contrast microscopy in living XTH-cells (Xenopus laevis tadpole-heart cells) in tissue culture. The same organelles have been viewed subsequently in electron micrographs. Locomotion of mitochondria proceeds at velocities up to 100 μm/min. Formation of branches of mitochondria and other shape changes may occur with the same speed. Mitochondrial motility can be classified into 4 types: (1) Alternating extension and contraction at the two ends of rod-shaped mitochondria. (2) Lateral branching. (3) Alternate stretching and contraction of arbitrary parts of a mitochondrion amounting to a kind of peristaltic action. (4) Transverse wave propagation along the organelle. Types 1 to 3 can be reduced to the same underlying principle; they cause locomotion. Formation of mitochondrial extensions is due to elongation of cristae. The observations are discussed in terms of 4 specific proposals. (1) Intracellular locomotion of mitochondria is caused by local enlargements and contractions of the organelles. (2) The shape changes are correlated with alterations in the arrangement of the cristae. (3) Such arrangements are not associated with overall swelling or shrinkage of the mitochondrion; they are local features. (4) Estimates of the time required for rearrangement of the inner compartment amount to less than 0·3 s for single crista arrangements during the fastest shape changes, and less than 1–3 s during slower alterations. This high velocity is in good accord with the hypothesis of energy conservation by conformational events during oxidative phosphorylation.

The antimitotic drugs colchicine and vinblastine were applied to the melanophores of isolated scales of Pterophyllum scalare. The time course of inhibition of pigment migrations was studied quantitatively with the photoelectric method.

A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts.

Summary Changes in the microcirculation of the mesentery of rabbit after ischemia of 45 min duration are demonstrated by capillary cinematography. Diapedesis of red blood cells, interstitial edema, and swelling of endothelial cells in the postcapillary vessels are the most frequent indications of damage observed. Intravenous injection of black ink can be used to show that the damage to endothelial cells starts in the venules. Most of the capillary lesions can be avoided by intravenous injection of Trasylol® (100000 KIU) before the application of a tourniquet. Almost the same effect is achieved if Trasylol® is injected 15 min before release of the circulation.