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New data on tooth enamel and dentin microstructure, obtained by acoustic microscopy methods, which are based on evaluation of distribution of the local elastic and mechanical properties, are presented. The results demonstrate that the acoustic microscopy methods are more sensitive to changes in mechanical properties of dental tissues in caries in comparison with optic microscopy.
A powerful new method is used to investigate the correlation between light microscopic and acoustic properties of biological tissues. Specimens of liver were sectioned into successive slices, 250 micrometers and 10 micrometers thick. The thick sections were investigated acoustically, the thin sections by means of light microscopy. Markers that could be detected and located, both optically and acoustically, were used to find and reconstruct corresponding regions in the acoustic and optical sections (2.5 x 2.5 mm). Parameter images were reconstructed from the sections investigated acoustically. The acoustic parameters were attenuation at 30 MHz, the slope of the attenuation spectrum (between 10 and 50 MHz), backscattering at 30 MHz, the slope of the backscattering spectrum (between 10 and 50 MHz) and the local ultrasound velocity. Acoustic images were obtained in the frequency range from 10 to 50 MHz, yielding a lateral resolution of about 50 micrometers. The sections for light microscopy were stained according to the Goldner trichrome staining technique. The histological composition was determined quantitatively, using digital image segmentation techniques. The percentage of collagen-rich fibrous tissue, luminal structure and interstitial spaces, and the number of nuclei were calculated for regions of 250 x 250 micrometers. These histological features were correlated with the acoustic parameters obtained from the corresponding regions in adjacent sections. It was thus possible to find the histological components responsible for acoustic parameters.
Several investigators have demonstrated the feasibility of imaging at the cellular level using acoustical microscopy. It has also been proposed that acoustical microscopy technology might be adopted for in vivo applications. Before such applications are implemented, it is important to demonstrate that any major deleterious effects are highly unlikely. To this end, we have repeatedly scanned NIH/3T3 mouse fibroblasts in culture using an Olympus UH3 acoustical microscope operating at 600 MHz. No adverse effects were observed even after exposures for 1 h. Spatial peak temporal averaged intensities were estimated to be below 300 mW/cm2.
Time-resolved adsorption behavior of a human immunoglobin G (hIgG) protein on a hydrophobized gold surface is investigated using multitechniques: quartz crystal microbalance/dissipation (QCM-D) technique; combined surface plasmon resonance (SPR) and Love mode surface acoustic wave (SAW) technique; combined QCM-D and atomic force microscopy (AFM) technique. The adsorbed hIgG forms interfacial structures varying in organization from a submonolayer to a multilayer. An "end-on" IgG orientation in the monolayer film, associated with the surface coverage results, does not corroborate with the effective protein thickness determined from SPR/SAW measurements. This inconsistence is interpreted by a deformation effect induced by conformation change. This conformation change is confirmed by QCM-D measurement. Combined SPR/SAW measurements suggest that the adsorbed protein barely contains water after extended contact with the hydrophobic surface. This limited interfacial hydration also contributed to a continuous conformation change in the adsorbed protein layer. The viscoelastic variation associated with interfacial conformation changes induces about 1.5 times overestimation of the mass uptake in the QCM-D measurements. The merit of combined multitechnique measurements is demonstrated.
Mechanical failure of the fibrous cap of a vulnerable atherosclerotic plaque may lead to sudden plaque rupture and thus precipitate arterial thrombosis. Because ultrasound correlates strongly with mechanical features of tissues it might provide information on the stability of fibrous caps. The acoustic properties of the normal vessel wall and plaques, particularly fibrous caps of lipid-rich plaques, were evaluated in the aortic roots of six normal C57BL mice and 12 atherosclerosis-prone apoE-deficient (apoE(-/-)) mice by scanning acoustic microscopy (SAM). After processing, the attenuation of high-frequency (1.1 GHz) focused ultrasound was measured in unstained tissue sections by SAM followed by quantification of the amount and type of collagen in picrosirius red stained sections by means of polarized light microscopy (PLM). The acoustic and optical images were superimposed and ultrasonic attenuation was measured in different tissue components. Pertinent plaque features, particularly fibrous caps covering lipid-rich pools, were clearly visualized by both SAM and PLM. Collagen appeared green in thin fibrous caps and bright orange in thick caps by PLM. The attenuation of ultrasound was significantly higher in the collagen fibers with orange color compared to those with green color (17.2 versus 6.6 x 10(3) dB/mm). SAM has shown the possibility to characterize the types of collagen by high frequency intravascular ultrasound in vivo and it might improve our understanding of the vulnerable plaque and its sudden rupture from micro-mechanical point of view.
Bone is a primary target for colonization of metastatic breast cancer cells. Once present, the breast cancer cells activate osteoclasts, thereby stimulating bone loss. Bone degradation is accompanied by pain and increased susceptibility to fractures. However, targeted inhibition of osteoclasts does not completely prevent lesion progression, nor does it heal the lesions. This suggests that breast cancer cells may also affect osteoblasts, cells that build bone. The focus of this study was to determine the ability of breast cancer cells to alter osteoblast function. MC3T3-E1 osteoblasts were cultured with conditioned medium from MDA-MB-231 breast cancer cells and subsequently assayed for changes in differentiation. Osteoblast differentiation was monitored by expression of osteocalcin, bone sialoprotein and alkaline phosphatase, and by mineralization. Osteoblasts cultured with MDA-MB-231 conditioned medium did not express these mature bone proteins, nor did they mineralize a matrix. Inhibition of osteoblast differentiation was found to be due to transforming growth factor beta present in MDA-MB-231 conditioned medium. Interestingly, breast cancer conditioned medium also altered cell adhesion. When osteoblasts were assayed for adhesion properties using interference reflection microscopy and scanning acoustic microscopy, there was a reduction in focal adhesion plaques and sites of detachment were clearly visible. F-actin was disassembled and punctate in osteoblasts cultured with MDA-MB-231 conditioned medium rather than organized in long stress fibers. Taken together, these observations suggest that metastatic breast cancer cells alter osteoblast adhesion and prevent differentiation. These affects could account for the continued loss of bone after osteoclast inhibition in patients with bone-metastatic breast cancer.
Bone attachment to two classes of hydroxyapatite (HA) coated polymers was evaluated mechanically and histologically. Particulate HA was molded into the surface of thermoplastic implants and cast into the surface of thermoset implants. Coated and uncoated implants of both types were implanted in the distal femurs of rabbits for four and twelve weeks. After sacrifice, the bone/implant interface was evaluated mechanically via a push-out test with a servohydraulic test system. Paired statistical analysis revealed significantly greater shear strengths for coated vs. uncoated implants for both polymer systems at both time periods. Mixed mode of failure occurred with particle removal from the polymer surface and from the bone. One animal at each time period was saved for histology. Histology at both time periods indicated direct bone apposition to the HA coating as compared to a fibrous encapsulation about the uncoated implants. This was confirmed with backscatter scanning electron microscopy and scanning acoustic microscopy. Hydroxyapatite coatings significantly improve the bond strength between polymers and bone by allowing direct bone apposition and some mechanical interlocking with the bone.
Measurement of the elastic properties of single osteon lamellae is still one of the most demanding tasks in bone mechanics to be solved. By means of site-matched Raman microspectroscopy, acoustic microscopy and nanoindentation the structure, chemical composition and anisotropic elasticity of individual lamellae in secondary osteons were investigated. Acoustic impedance images (911-MHz) and two-dimensional Raman spectra were acquired in sections of human femoral bone. The samples were prepared with orientations at various observation angles theta relative to the femoral long axis. Nanoindentations provided local estimations of the elastic modulus and landmarks necessary for spatial fusion of the acoustic and spectral Raman images. Phosphate nu(1) (961 cm(-1)) and amide I (1665 cm(-1)) band images representing spatial distributions of mineral and collagen were fused with the acoustic images. Acoustic impedance was correlated with the indentation elastic modulus E(IT) (R(2)=0.61). Both parameters are sensitive to elastic tissue anisotropy. The lowest values were obtained in the direction perpendicular to the femoral long axis. Acoustic images exhibit a characteristic bimodal lamellar pattern of alternating high and low impedance values. Since this undulation was not associated with a variation of the phosphate nu(1)-band intensity in the Raman images, it was attributed to variations of the lamellar orientation. After threshold segmentation and conversion to elastic modulus the orientation and transverse isotropic elastic constants were derived for individual ensembles of apparent thin and thick lamellae. Our results suggest that this model represents the effective anisotropic properties of an asymmetric twisted plywood structure made of transverse isotropic fibrils. This is the first report that proves experimentally the ability of acoustic microscopy to map tissue elasticity in two dimensions with micrometer resolution. The combination with Raman microspectroscopy provides a unique way to study bone and mineral metabolism and the relation with mechanical function at the ultrastructural tissue level.
In vivo ultrasound examination has been introduced into dermatology as a non-invasive diagnostic technique. The resolution capacity, however, has been limited so far, and the in vivo ultrasonic features have not been fully understood. Using high frequencies (GHz range) in acoustic microscopy, we have been able to achieve a resolution comparable to that in light microscopy. In a first step, we examined normal human skin sections and compared them to histological specimens. The following study on melanocytic nevi, malignant melanoma, and metastases of melanoma showed that their acoustic images were more pronounced than those of the surrounding tissue. The echo intensity of solid basal cell carcinoma and squamous cell carcinoma resembled that of the epithelium. The structures found in acoustic microscopy can be correlated to those seen in light microscopy.
Application of a line-shaped point spread function (PSF) to imaging of void defects in directly bonded wafers is considered. Two non-confocally adjusted spherical transducers are employed to implement an acoustic microscope operating in transmission with a time dependent point spread function, whose shape is optimized by both temporal apodization of the received signal and spatial apodization of the transducer aperture. Strong imaging artifacts resulting from the generation and detection of edge waves are eliminated in this way. It is shown by several examples that only a broadband system can be utilized in order to obtain a line-shaped PSF suitable for imaging.
Acoustic microscopy permits a native, nondestructive and artefact-free analysis of the surface of intraocular lenses with regard to manufacturing defects, biologic cellular and membranaceous layers, and signs of degradation. Furthermore, it enables structures below the surface to be evaluated, so that inclusions, pores, microcracks and zones of deformation can be detected. In addition to performance of a one-dimensional, relative intensity profile of the surface and the corresponding pseudo-3D image, the properties of the materials inside the intraocular lens can be characterized by computerized analysis of the reflected ultrasound energy.
Acoustic microscopy (30-60 microm resolution) and nanoindentation (1-5 microm resolution) are techniques that can be used to evaluate the elastic properties of human bone at a microstructural level. The goals of the current study were (1) to measure and compare the Young's moduli of trabecular and cortical bone tissues from a common human donor, and (2) to compare the Young's moduli of bone tissue measured using acoustic microscopy to those measured using nanoindentation. The Young's modulus of cortical bone in the longitudinal direction was about 40% greater than (p<0.01) the Young's modulus in the transverse direction. The Young's modulus of trabecular bone tissue was slightly higher than the transverse Young's modulus of cortical bone, but substantially lower than the longitudinal Young's modulus of cortical bone. These findings were consistent for both measurement methods and suggest that elasticity of trabecular tissue is within the range of that of cortical bone tissue. The calculation of Young's modulus using nanoindentation assumes that the material is elastically isotropic. The current results, i.e., the average anisotropy ratio (E(L)/E(T)) for cortical bone determined by nanoindentation was similar to that determined by the acoustic microscope, suggest that this assumption does not limit nanoindentation as a technique for measurement of Young's modulus in anisotropic bone.
Targeted acoustic contrast agents offer the potential for sensitive ultrasonic detection of pathologic tissues. We have previously reported the development of a ligand-targeted, lipid-encapsulated, liquid perfluorodichlorooctane ultrasonic contrast system with a small nominal particle size (approximately 250-nm diameter). Perfluorocarbon nanoparticles substantially increase reflectivity when bound to targeted surfaces, and we propose that this system can be approximated physically as a simple, thin layer, acoustic transmission line. In this study, we evaluate this model and compare the ultrasonic reflectivity of different perfluorocarbon formulations with widely varying acoustic impedances targeted to either nitrocellulose membranes or plasma thrombi in vitro. Five perfluorocarbons were investigated: perfluorohexane (PFH), perfluorooctane (PFO), perfluorooctyl bromide (PFOB), perfluorodichlorooctane (PFDCO), and perfluorodecalin (PFD). Ultrasonic reflection was measured by acoustic microscopy (17 to 35 MHz). Acoustic reflectivity was increased (P < 0.05) by all targeted perfluorocarbon formulations, and the magnitude of the contrast effect was inversely correlated with the perfluorocarbon acoustic impedance. PFH nanoparticles exhibited the greatest enhancement, and PFD nanoparticles showed the least. The acoustic transmission line model predicted well the relative differences in acoustic reflectivity and frequency dependence among the perfluorocarbon formulations. For future clinical applications, PFO nanoparticles may provide the best combination of acoustic enhancement, in vivo physical stability, and safety.
The acoustic microscopy technique provides some extraordinary advantages for determining mechanical properties of living cells. It is relatively fast, of excellent spatial resolution, and of minimal invasiveness. Sound velocity is a measure of the cell stiffness. Attenuation of cytoplasm is a measure of supramolecular interactions. These parameters are of crucial interest for studying cell motility and volume regulations and to establish the functional role of the various elements of the cytoskeleton. Using a scanning acoustic microscope, longitudinal wave speed, attenuation and thickness profile of a biological cell were measured earlier by Kundu et al. [Biophys. J. 78, 2270-2279 (2000)]. In that study it was assumed that the cell properties did not change through the cell thickness but could vary in the lateral direction. In that effort the acoustic-microscope-generated signal was modeled as a plane wave striking the cell at normal incidence. Such assumptions ignored the effect of cell inhomogenity and the surface skimming Rayleigh waves. In this paper a rigorous lens model, based on the DPSM (distributed point source method), is adopted. For the first time in the literature the cell is modeled here as a multi-layered material and the effect of some external drug stimuli on a living cell is studied.
Since a valid macroscopic investigation fundamentally cannot exist without its basis in microscopy, we are seeking to identify characteristic echo structures by means of acoustical microscopy at frequencies of 600 MHZ and 1 GHZ in order to better understand ultrasonography in everyday practice and to determine reliable and specific imaging characteristics of the different components of breast tissues. We have studied slices of mammary tissue 5-10 microns thick which had been removed during surgery. Acoustical imaging has to be performed in transmission mode as well as reflection mode. The intrinsic acoustic contrast is more than sufficient to obtain good visualization of different tissue components of the mammary gland. We noted, however, that even if the microscopic acoustic images were of good quality--often as good as light microscopy--certain tissue components such as the epithelium are irregularly visualized. It appears that the effective visualization of a structure depends entirely upon this acoustical environment.
The ultrasonic methods, which detect applied or residual stress in materials, are based on nonlinear interaction of a small dynamic disturbance (acoustic waves) with the pre-deformed state of the solid. This weak phenomenon (acoustoelasticity) leads to a dependence of acoustic wave velocities on the initial stress, and a stress-induced anisotropy in the acoustical properties of the material. In anisotropic media, the transversal wave velocity depends on its polarization. The amplitude of the conical polarized shear wave, propagating through a plate specimen, is sensitive to pre-stress due to acoustoelastic birefringence. The resulting scan image is created by variations of the amplitude. The previous description is a basic principle of the approach used for stress mapping in Al-alloys by time-resolved acoustic microscopy. Disk specimens with central stress concentrators are loaded step by step. The acoustic scans are created during each loading step. Thermal stress detection is also shown on specimens with an Invar core. The original image processing procedure has been developed to improve edge detection of obtained stress maps. The acoustic images are compared with theoretically predicted isocline contours. The inherent material anisotropy and the structural inhomogeneities influence significantly the acoustoelastic measurements. Advantages and limitations of the nondestructive technique are summarized on the basis of presented experimental results.
Scanning acoustic microscopy (SAM) was used in the burst mode at 400 MHz, nominal lateral resolution 2.5 microm, to study the micromechanical properties of the dentin/adhesive interface. Corresponding specimens from the same tooth were investigated using mu Raman spectroscopy, light microscopy, and scanning electron microscopy.12 The elastic moduli of the components of the dentin/adhesive interface were determined by comparing the recorded acoustic impedance values to a calibration curve generated on standard materials. The standard materials, which include polypropylene, Teflon, PMMA, pyrex glass, aluminum, titanium, and stainless steel, provide the appropriate range of acoustic impedance values. The elastic moduli of the components of the dentin/adhesive interface are: partially demineralized dentin, 13 Gpa; mineralized dentin, 28 GPa; adhesive, 5.0 GPa; and unprotected protein at the interface < 2.0 GPa.
In this study the influence of various histological techniques on the acoustic parameters of liver tissue was investigated. Radiofrequency (RF) echographic data were obtained in vitro from 21 liver samples taken from 8 white New Zealander rabbits. The samples were measured in four different subsequent histological tissue processing conditions (freshly excised, 4% buffered formalin fixed, after it went through a paraffin cycle and after staining with hematoxylin and eosin). The acoustic parameters that were obtained from the rf data were velocity of sound, slope of the attenuation coefficient versus frequency between 1.9 and 6.9 MHz, attenuation coefficient at 4.4 MHz, slope of the backscattering spectrum between 1.9 and 6.9 MHz, and intercept of the backscattering spectrum. It was found that fixation by formalin preserves the acoustic properties of the tissue to a reasonable extent. Embedding in paraffin and deparaffinizing induces large changes in the acoustic properties of the tissue. As an alternative, freezing prior to cutting, rather than the paraffin cycle, was investigated also in 10 liver samples obtained from 4 New Zealander rabbits. This method produced no significant changes of the acoustic parameters and should therefore be preferred in acoustic microscopy.