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The preservation of mitochondria, cytoplasmic vacuoles and cytoplasm by various fixatives after various pretreatments of ethothelial heart cells from Xenopus laevis tadpoles in tissue culture was investigated. The study was based on phase contrast cinemicrographic recordings and on qualitative and quantitative observations with the electron microscope. Three fixatives were used: 3% glutaraldehyde in phosphate buffer, followed by 1% osmium tetroxide postfixation, fixation only with 1% osmium tetroxide in phosphate buffer and the fixing medium according to Dalton. Cells were either not treated or pretreated for 20 min: 10 microM FCCP (Carbonylcyanide-p-trifluoromethoxy-phenylhydrazone) or 4 mM KCN. The superiority of glutaraldehyde was exemplified by its very rapid action, good preservation of cytoplasm, vacuoles, and mitochondria. It was the only medium which maintained an electron density of the mithochondria matrix. In both of the other fixatives swelling of mitochondria and coagulated appearance of cytoplasm (in phase contrast) was more pronounced in cells pretreated with metabolic inhibitors than in controls. Observations with the light microscope have been confirmed by morphometry of electron micrographs of mitochondria. The relation of matrix space to intracristal space is changed in opposite directions after glutaraldehyde and after the Dalton-type fixation. The results indicate a higher sensitivity against fixation artifacts in cells under pathological conditions than normal cells.

One of the most important problems in automatic image analysis is the discrimination of features by, a certain range of grey levels. To obtain as many as possible different grey levels sufficient contrast is achieved mainly by specific staining. Alternative and supplementary methods are some micrscopical methods, not routinely used. Interference microscopy, interference reflexion microscopy and microfluorimetry are discussed in detail. These optical procedures enhance the contrast of specimen specifically without the necessity for the application of specimen specifically without the necessity for the application of sophisticated staining methods. In interference contrast, tissues can be separately detected by grey level discrimination due to varying concentration of dry mass; this is shown for a cornifying part of fish skin (breeding tubercle of Rutilus rutilus L.). Furthermore, very small amounts of dry matter can be determined with high precision, as demonstrated for a single tissue culture cell (XTH-cell). Automatisation of image analysis provides a unique opportunity for routine application of interferometric measurements. The principles of the procedures are outlined. By interference reflexion microscopy cellular attachment areas to a glass surface are visualized, providing a powerful tool in cellular diagnosis based on grey level discrimination (darkest parts correspond to zones of closest contact to the substratum). A fast migrating lymphocyte and stationary endothelial cells have been chosen for demonstration. Various histochemical problems can be solved elegantly by fluorescence methods, e.g. mitochondria in living cells are specifically stained by a fluorochrome (DASPMI) and the distribution of fluorescence intensity can be followed within the mitochondrial population of a cell. Fluorescence was recorded from fotographic negatives taken with a fluorescence microscope. Additionally a short comment is given on the application of polarisation microscopy for feature detection.

A population of endothelial cells grown 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 microm2) 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.

Sequestration of iontophoretically injected Ca2+ by monolayer culture cells (primary Xenopus laevis Tadpole Heart cells, XTH P, and an established cell line, XTH 2) is investigated. Injections are made at different velocities by changing the influx current. On Ca2+ injection the entire ER desintegrates, and near to the tip of the injecting pipette microtubules depolymerize. The time required to attain cell death is taken as the parameter indicating an overload of cellular Ca2+ sequestration capability. Three different Ca2+ transport kinetics are found: at Ca2+ flux rates of up to 20 X 10(-15) mol X s-1 (condition I) cells can tolerate long injection periods before they die; at flux rates from 20 to 40 X 10(-15) mol X s-1 (condition II) the injection time before cell death remains constant. Flux rates exceeding 40 X 10(-15) mol X s-1 decrease cellular Ca2+ sequestration capability to a minimum. These observations support the assumption of two Ca2+ sequestrating mechanisms: one of high affinity, but with low capacity (less than = 5 X 10(-15) mol X s-1) the other with low affinity for Ca2+ and a high capacity (10 to 40 X 10(-15) mol X s-1) for Ca2+ accumulation. Both mechanisms are saturable. As the Ca2+ sequestration velocity remains approximately constant in condition II, the capacity of the second mechanism seems to grow with increasing Ca2+ influx. The highly affin Ca2+ compartment is the ER, mitochondria form the less affin system. XTH 2 differ from primary cells by possessing a 5 to 8 fold higher Ca2+ sequestration capacity, whereas sequestration velocity is equal in both cell types.

Reflection contrast microscope methods are generally used for studies of those portions of the cell that are turned towards the glass coverslip, to comprehend the structure of the cytoskeleton and the dynamics of cell movement, as well as formation of cell-glass contacts. In incident illumination only reflected light contributes to picture formation. The intensity of which in the case of observation of unstained cells is small because of small refraction differences. To overcome this problem a reflection contrast system was developed by Leitz according to Ploem [49], in which by using contrast preserving measures the reflection becomes prominent in comparison with the lens reflexes. The emerging pictures are a result of interferences of reflections at glass-cell, cell-culture medium and culture medium-cell interfaces. According to Fresnel's equations the reflected intensity depends on the differences of the particular refractive indices and the thickness of the layers, which determine the phase of interfering beams. In idealized systems of thin films the reflected intensity is a measure for their optical constants. Relative reflection measurements from glass-cell areas is comparison with the known glass-medium reflection, can therefore be revealing as far as refraction index, cell-glass distance or cell thickness are concerned. The estimates by Bereiter-Hahn et al. [15] were made in the assumption of vertical illumination neglecting its actual conical shape: the comparison of two Fresnel functions of cytological relevant measurements show - in accordance with Gingell and Todd [24] - that this is only permitted under certain conditions, depending on the required accuracy of the measurements; an incidence angle of about 30 degrees leads to an error of about 10%, an angle of 50 degrees to more than 50%.

In XTH-2 cells, a line derived from tadpole heart endothelia, changes of the F-actin pattern have been followed after injection of calcium and after permeabilization of the cells to calcium using the ionophore A 23187 and an external pCa of 6.0. On the dorsal surface numerous microvilli appear, the stress fibres show an interrupted pattern and F-actin accumulates at the cell periphery. These reactions are interpreted to result from contractions. Finally most of the F-actin becomes depolymerized (in about 30 min). In addition actin containing accumulations are developed in the perinuclear area. In G1 cells stress fibres are less sensitive to enhanced calcium, they persist for more than 30 min in pCa 6.0. Taking into account a relatively high cytosolic calcium concentration in many malignantly transformed cells, their more rounded appearance and lack of an ordered array of actin fibrils could be closely related phenomena.

On reoxygenation of ischemic or hypoxic hearts a sudden release of cytosolic enzymes coupled with hypercontraction and cell injury occurs, which has been termed the "oxygen paradox". We have attempted to imitate this phenomenon in cultured chick myocytes to try to find the cause of this sudden enzyme release. During 4 hours of normoxic perfusion (pH 7.4) monolayer cultures of chick embryonic myocytes retain their normal morphology, beat rhythmically, and show no release of creatine kinase (CK) into the perfusate. Hypoxic perfusion (O2 less than or equal to 0.25 microliter/ml) stops cell contraction (15-20 min) and causes "blebbing" of the sarcolemma (20-30 min). Membrane blebs increase in size and number with continuing hypoxia and eventually the cells become irreversibly damaged. Perfusion at pH 7.4 leads to a release of CK shortly after membrane damage occurs (30-40 min), with peak enzyme levels at 60-90 min. Reoxygenation after 120 min hypoxia does not exacerbate release. Hypoxic perfusion at pH 7.0 suppresses the release of CK from the cells despite extensive membrane blebbing. Normoxic perfusion at pH 7.4 after 100 min hypoxia (pH 7.0) causes an efflux of enzyme from the irreversibly injured cells. This can be prevented by reoxygenating the cells at pH 7.0 and stimulated by raising the pH of the hypoxic perfusate to 7.4. Shorter hypoxic periods (30 mins) at pH 7.0 followed by normoxic perfusion at pH 7.4 lead to a sudden large efflux of CK, arrhythmic contractions and hypercontraction of myofilaments, i.e. the typical symptoms of the "oxygen paradox". Thus changes in external pH can influence the release of intracellular enzymes during hypoxia and reoxygenation.

The reactions of XTH-2 cells (line derived from Xenopus laevis tadpole hearts) to cytochalasin D (CD) were followed using scanning acoustic microscopy (SAM) at 0.9 GHz, fluorescence and electron microscopy. The first reaction to CD which can be detected by SAM is a loss of image contrast, indicating a decrease in acoustic impedance of about 30%. Based on structural changes revealed by staining of actin with TRITC-phalloidin, and taking theoretical considerations into account, a relationship between impedance decrease and tension in the actin fibrillar system is deduced.

FITC-Con A-fluorescence and electron microscopy have been used in parallel for the visualization of rough endoplasmic reticulum in Xenopus 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.

An investigation has been made on the properties of dimethylaminostyrylmethylpyridiniumiodine (DASPMI), its reaction with isolated pigeon heart mitochondria and its suitability as a vital strain for mitochondria in situ. DASPMI is a low toxicity specific vital stain for mitochondria in living cells. In vitro dye concentrations over 6 nmol/mg protein inhibit fast (state 3) respiration after a preincubation time of more than 5 min in the presence of substrate. No uncoupling was observed. Energization of pigeon heart mitochondria by addition of ATP or various substrated yields an average 8.5-fold increase in fluorescence intensity in relation to DASPMI-stained mitochondria that are under anoxia, substrate deficiency, or under the influence of respiratory inhibitors, or uncouplers. The alterations in fluorescence intensity are not primarily due to ion movements or pH changes. The amount of dye (2.96+/-0.8 nmol) yielding maximal fluorescence response with 1 mg mitochondrial protein remains constant during energization of mitochondria. As indicated by electron microscopic studies the observed changes in emission intensity may be related to changes in the fine structural organisation of cristae. A remarkable difference exists between isolated mitochondria and mitochondria in situ with respect to the reaction to cyanide. According to the reported results DASPMI will be a useful probe for the investigation of mitochondrial activities in living cells.

The role of calcium in the induction of locomotion, control of direction of locomotion, and modulation of shape of epithelial cells derived from Xenopus laevis tadpole epidermis is investigated. Local influx of calcium is achieved by electrophoretic release of small amounts of calcium from a micropipette (tip diameter 0.1-0.5 micron) closely apposed to the cell body or lamella. The cells are made permeable for calcium by calcium ionophore A23187, and they are kept in Ca++-free, Mg++-rich EGTA Ringer. Another method used to induce Ca++ influx is local application of A23187 while cells move in normal culture medium. Influx of Ca++ into the lamella induces a localised increase in thickness and enlargement of the lamella. Stationary cells become active and show movement in the direction of the Ca++ gradient. Fried-egg-shaped cells tend to acquire a semicircular shape and start moving. Moving cells change the direction of their locomotion, following the direction of Ca++ release. Influx of Ca++ in the cell body region induces its contraction concomitant with an increase in lamellar area. These observations suggest the presence of two different Ca++-sensitive components: an actomyosin meshwork in the cell body and an actin gel in the lamella. Influx of Ca++ induces contraction of actomyosin and solation of actin gel. Interaction of these two systems would explain modulation of shape and generation of locomotion in epithelial cells.

Endothelial cells derived from Xenopus tadpole hearts (XTH-2: Schlage et al. 1981) have been permeabilized for calcium using the ionophore A 23187. A Ca2+-concentration of 1 microM causes a graded disassembly of microtubules (mts): with increasing periods of incubation mts disintegrate and then depolymerize completely. The frequency of the different stages of depolymerization depends on the length of the Ca2+ incubation period, even after 60 min incubation time some mts remain. The stage of depolymerization of mts in a cell is correlated with its cell cycle phase showing the highest stability during S phase.

Dependence of locomotion of Xenopus laevis epidermal cells on calcium influx from the external medium was investigated. Inhibition of Ca2+ influx by 2 mM La3+ or 4 mM Tb3+ in the culture medium causes an immediate stop to locomotion and a loss of motion at the outer margin of the lamella; microcolliculi disappear and the entire lamella becomes flat and very thin. The cell body region enlarges by spreading into the lamella to an extent approximately coincident with the distribution of myosin. The increase in thickness of this area is the result. The cytoskeletal elements actin, alpha-actinin and myosin become homogeneously distributed throughout the cell and a great number of straight microtubules extend to the margin after 20 min in La3+-containing media. Prekeratin distribution does not change. Reduction of calcium concentration in the external medium by EGTA leads to cessation of cell locomotion. Sr2+ (1-4 mM) is also able to replace calcium for triggering locomotion. These findings point to a control of Ca2+-activated contractions of actomyosin by influx of external Ca2+. According to our model of cell locomotion [14] the contractions generate a hydrostatic pressure extending the lamella by flow of hyaloplasm towards the margin. Small swellings (microcolliculi) appearing thereby will be dislocated by a calcium-dependent sol-gel transformation in this area, which contains actin but not myosin.

The migration of keratocytes isolated from Xenopus tadpole epidermis has been investigated in vitro. In saline the cells move with a mean speed of 5-6 microns/min. Migration is slowed down in saline with diminished sodium content and ceases in media containing not more than 4 mM sodium. Inhibition of the Na+/K+-2Cl- cotransporter by piretanide reduces the speed of migrating cells to about one-third of the control level, the same accounts to inhibition of the Na+/H+ antiport with amiloride at pH 7.2. At pH 6.6, however, amiloride only slightly influences locomotion. Depolarization of the plasma membrane by increased extracellular K+ concentration or by inhibition of the Na+/K+ pump by ouabain is only of minor influence during more than 1 h. Hyperpolarization of the cells using the sodium ionophore monensin impedes locomotion; this inhibition depends on an active Na+/K+ pump. Ionophore-mediated breakdown of the K+ gradient strictly inhibits locomotion. The experiments have shown that a continuous flux of sodium ions is indispensable for the maintenance of cell locomotion. These ions may exert their action primarily by affecting cytosolic free calcium concentration and pH.

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 degrees; 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.

The formation of focal contacts by fibroblast-like 3T3 and 9390 cells and endothelial XTH-2 cells was examined with the use of reflection interference contrast microscopy at different cell densities. It was found that cells density-inhibited in growth loose all focal contacts with the substratum. This observation explains the disappearance of stress fibers in density-inhibited cells, and also offers an explanation for the mechanism of density-dependent inhibition of proliferation of anchorage-dependent cells.

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 mup. 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 or EGTA 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. It is concluded that a state of equilibrium exists between the external force and an opposing force produced by the melanophore. The differences in the opposing force under different experimental conditions may be due to a "contractile component". This component seems to be independent of microtubules, as indicated by vinblastine experiments. It "contracts" under aggregating stimulus and "relaxes" under dispersing stimulus. From the data presented in this paper, the order of magnitude of the pressure developed by the contractile component in the completely aggregated state was calculated as between 5-7 p/cm2 in the relaxed state and about 20 p/cm2 during contraction. These values are comparable to those observed in other nonmuscular cells.

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.

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 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 relative content of F-actin has been determined for three established cell lines, an amphibian cell line XTH-2 of endothelial origin, 3T3 cells and SV 40 transformed 3T3 cells. In the two non transformed lines F-actin decreases with increasing cell density starting with the onset of confluency of the culture. SV 40 transformed 3T3 cells generally contain less F-actin and do not show any significant point of change. The decrease in F-actin with increasing cell density is accompanied by a disappearance of stress fibres. SV 40 3T3 cells generally are devoid of stress fibres. The observations are discussed considering a possible involvement of F-actin in growth control.