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Lead Zirconate Titanate (PZT) transducers are commonly used to generate guided acoustic waves for health monitoring of structures made of isotropic and anisotropic materials. Therefore, characterization of PZT transducers is very important not only for reliability but also for the accuracy of data. In this research, Scanning Acoustic Microscopy (in reflection mode) at 30 MHz excitation frequency is conducted for determining the quality of disc type guided wave transducers. Multiple samples are scanned but only four transducers are selected for further investigation. Guided waves are dispersive in nature and multiple guided wave modes can coexist at single frequency. It has been already reported in the literature that good bonding condition between transducer and testing material is very important to restrict other factors that can affect the strength of recorded signals. Chirp signal is excited in the specimen in a single sided excitation/detection setup to investigate the effects of transducer quality on monitoring plate like structures. Sideband peak count technique is used for comparative study of transducers.

Ultrasound (US) is a widely used modality for medical imaging, since it is non-invasive and relatively inexpensive. The ability of US imaging to detect internal structures, like tissue interfaces or lesion sites makes it a promising candidate for dental imaging. So far, the inherent technical difficulties of US imaging in tissues and organs with very heterogeneous (acoustic) properties and limited access have prevented its widespread use. In this study, we characterized the acoustic properties of sectioned teeth by scanning acoustic microscopy in reflection and transmission modes. The spatial distribution of sound velocity was measured in sections of extracted human teeth by use of a scanning acoustic microscope (SAM). Freshly extracted teeth were fixed in 4% formaldehyde solution and embedded in a polymer block (PMMA). Sections of approximately 1 mm thickness were cut along the coronal-apical axis. Radio frequency (RF) data of teeth were collected in a scan region of 15 × 15 mm 2 by a SAM operating at 30 MHz with a lateral step size of 50 µm and a sampling rate of 500 MSa/s. Sound velocity was determined from the time resolved reflection and transmission signals. Values for sound velocity from transmission mode were about 20% lower than that from the reflection mode, if thickness information from reflection mode was used. If thickness was determined from the transmission mode, sound velocities from transmission were very close to those obtained from the reflection mode. Transmission mode is less sensitive to artifacts caused by the inclination of the specimen.

G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress and treatments with 12-O-tetradecanoylphorbol-13-acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM-G3 cells. Elevated invasiveness of G3S1 cells was accompanied by higher incidence of myzitiras morphotype (sucker-like) and newly observed vthela morphotype (leech-like) both inducible in Hanks' Balanced Salt Solution test. Time-lapse phase contrast microscopy showed a capacity of G3S1 cells to form lobopodial protrusions already 20 min after seeding on gelatin. These protrusions could make contact with the dish and possibly produce the vthela shape. The possible relationship of mysitiras and vthela morphotypes to an increase in malignant potential marked by enhanced invasiveness was thus indicated. SCANNING 31: 102–106, 2009. © 2009 Wiley Periodicals, Inc.

Highly dynamic mitochondrial morphology is a prerequisite for fusion and fission. Mitochondrial fusion may represent a rescue mechanism for impaired mitochondria by exchanging constituents (proteins, lipids and mitochondrial DNA) and thus maintaining functionality. Here we followed for the first time the dynamics of a protein complex of the respiratory chain during fusion and fission. HeLa cells with differently labelled respiratory Complex I were fused and the dynamics of Complex I were investigated. The mitochondrial proteins spread throughout the whole mitochondrial population within 3 to 6 h after induction of cell fusion. Mitochondria of fused cells displayed a patchy substructure where the differently labelled proteins occupied separate and distinct spaces. This patchy appearance was already--although less pronounced--observed within single mitochondria before fusion, indicating a specific localization of Complex I with restricted diffusion within the inner membrane. These findings substantiate the view of a homogenous mitochondrial population due to constantly rearranging mitochondria, but also indicate the existence of distinct inner mitochondrial sub-compartments for respiratory chain complexes.

Tight regulation of the cell volume is important for the maintenance of cellular homeostasis. In a hypotonic environment, cells swell owing to osmosis. With many vertebrate cells, swelling is followed by an active reduction of volume, a process called regulatory volume decrease (RVD). A possible participant in RVD is the non-selective cation channel TRPV4, a member of the TRP superfamily that has been shown to react to hypotonic stimuli with a conductance for Ca2+. As a model for cell-volume regulation, we used a human keratinocyte cell line (HaCaT) that produces TRPV4 endogenously. When HaCaT cells were exposed to a hypotonic solution (200 mOsm) maximal swelling was followed by RVD. During swelling and volume regulation, a strong Ca2+ influx was measured. Gd3+, an inhibitor of TRPV4, blocked RVD of HaCaT cells and the accompanying rise of cytosolic Ca2+. To define the role of TRPV4 in volume regulation, a TRPV4-EGFP fusion protein was produced in CHO cells. CHO cells are unable to undergo RVD under hypotonic conditions and do not produce TRPV4 endogenously. Fluorescence imaging revealed that recombinant TRPV4 was localized to the cell membrane. Production of TRPV4 enabled CHO cells to undergo typical RVD after hypo-osmolarity-induced cell swelling. RVD of TRPV4-transfected CHO cells was significantly reduced by Gd3+ treatment or in Ca2+-free solution. Taken together, these results show a direct participation of TRPV4 in RVD.

As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

Combining the use of cells with sparse cristae marked with IMP–EGFP and short pulsed sub-saturating fluorescence excitation (non-saturation fluorescence microscopy/NSFM) revealed inhomogeneous fluorescence distribution along mitochondria in living cells. Also the matrix located TMRE was distributed non-uniformly and at least in part filling the gaps between the IMP–EGFP fluorescence: fluorescence intensities are modulated in space and time in part in an antidromic manner. The spatial modulations can be interpreted to represent cristae/matrix distributions. The temporal fluctuations of fluorescence vary within 0.3–3 s. Because most peak positions of IMP fluorescence remain stationary up to at least several minutes, temporal intensity modulations may result from varying emissions related to the degree of excitation and/or represent wobbling of cristae, i.e. lateral movements, bending or size changes. Modulations by noise and non-saturated excitation have been reduced by 3 steps of deconvolution followed by averaging 4 images. This allowed a final temporal resolution of 150 ms. Disappearance of cristae or formation of new ones takes place within a few seconds, but these are rare events. Thus position of cristae seems to be rather stable, but they regularly disassemble close to fission sites. Treatment with oligomycin strongly reduces “wobbling” activity.

G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress and treatments with 12-O-tetradecanoylphorbol-13-acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM-G3 cells. Elevated invasiveness of G3S1 cells was accompanied by higher incidence of myzitiras morphotype (sucker-like) and newly observed vthela morphotype (leech-like) both inducible in Hanks' Balanced Salt Solution test. Time-lapse phase contrast microscopy showed a capacity of G3S1 cells to form lobopodial protrusions already 20 min after seeding on gelatin. These protrusions could make contact with the dish and possibly produce the vthela shape. The possible relationship of mysitiras and vthela morphotypes to an increase in malignant potential marked by enhanced invasiveness was thus indicated.

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 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 scanning acoustic microscope, longitudinal wave speed, attenuation and thickness profile of a biological cell have been measured earlier by Kundu, Bereiter-Hahn and Karl (2000) from the voltage versus frequency or V(f) curves in the frequency range 980-1100 MHz. Two limitations of that study are overcome in this paper. In that study it was assumed that the cell properties did not change through the cell thickness and could vary only in the lateral direction. Secondly, the acoustic microscope generated ultrasonic signal was modeled in that study as a plane wave striking the cell and the substrate at normal incidence. Such assumption ignores the contribution of the surface skimming Rayleigh waves. Improved and more generalized analysis that is presented here avoids such restrictive assumptions. For the first time, in this paper the cell is modeled as a multi-layered material with different properties for nucleus and surrounding cell material. The inverse problem is solved to study the effect of drugs on living cells.

In the middle of the 90s, the image processing and analysing method subtraction scanning acoustic microscopy (SubSAM) was developed to display and quantify cell surface motility. Since then, the approach of this method supported already existing models and provided new insights into the regulation of cell surface motility. The investigations with SubSAM revealed that cell surface motility is modulated by tension and is regulated by known F-actin reorganising signal transduction pathways via the small GTP-binding protein Rac and protein kinase C. Beyond that, the in vitro cell surface motility as revealed by SubSAM corelates with cell transformation respectively the invasive or metastatic potential. The enhancement of cell surface motility is always accompanied by characteristic actin reorganisation: loss of stress fibers and the formation of cortical F-actin knots or larger aggregates. From these observations a model has been developed for the regulation of cell surface motility and an addtitional mechanism for cell surface deformation based on myosin-dependent F-actin aggregation is proposed.

The physical properties of cytoplasm are primarily determined by the state of cytoskeletal element, i.e. their polymerisation, crosslinking and supramolecular interactions with other molecules. These interactions are involved in signal transduction processes as well as in morphogenesis. Scanning acoustic microscopy proved to be a powerful tool to determine the mechanical properties of living cells. The interpretation of the sound propagation parameters, however, has to be based on investigation of in vitro models. Therefore polymerisation of actin and tubulin have been followed using a novel oscillating rod rheometer which allows for synchronous determination of sound velocity, sound attenuation and viscosity. Sound velocity measures the elastic propterties of cytogels, attenuation the supramolecular associations. All these parameters are evaluated with minimal strain, in the range of 1- 100 nm actin with glycolytic enzymes not only modulated polymerisation in a specific, and substrate dependent manner, but also the stiffness of the fibrils was altered, e.g. by hexokinase in the presence of high ATP, this enzyme exhibited actin severing properties and reduced stiffness. Differences in polymerisation kinetics were observed comparing pyrene-labeled actin fluorimetry and oscillating rod viscosimetry. This comparison led to the detection of pseudocrystalline structures produced by g-actin and aldolase (in the absence of fructose-bisphophate, the substrate of aldolase). Elastic stiffness of actin filaments can be modulated by ATP/ADP and by actin binding proteins (e.g. the glycolytic enzyme hexokinase) as well. The in vitro observations support the interpretation of SAM data calculated for living cells.

Polymerization and depolymerization of cytoskeletal elements maintaining cytoplasmic stiffness are key factors in the control of cell crawling. Rheometry is a significant tool in determining the mechanical properties of the single elements in vitro. Viscoelasticity of gels formed by these polymers strongly depends on both the length and the associations of the filaments (e.g. entanglements, annealings and side-by-side associations). Ultrasound attenuation is related to viscosity, sound velocity and supramolecular structures in the sample. In combination with a small glass fibre (2mm×50µm), serving as a viscosity sensor, an acoustic microscope was used to measure the elasticity and acoustic attenuation of actin solutions. Changes in acoustic attenuation of polymerizing actin by far exceed the values expected from calculations based on changes in viscosity and sound velocity. During the lag-phase of actin polymerization, attenuation slightly decreases, depending on actin concentration. After the half-maximum viscosity is accomplished and elasticity turns into steady state, attenuation distinctly rises. Changes in ultrasound attenuation depend on actin concentration, and they are modulated by the addition of α-actinin, cytochalasin D and profilin. Thus absorption and scattering of sound on the polymerization of actin is related to the packing density of the actin net, entanglements and the length of the actin filaments. Shortening of actin filaments by cytochalasin D was also confirmed by electron micrographs and falling-ball viscosimetry. In addition to viscosity and elasticity, the attenuation of sound proved to be a valuable parameter in characterizing actin polymerization and the supramolecular associations of F-actin.