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PubMed1995

The anisotropy of osteonal bone and its ultrastructural implications.

The anisotropic elastic symmetry of osteonal bone reflects the ultrastructural organization of collagen fibrils and mineral crystals within the osteons as well as the lamellar microstructure. Until recently, reported values for bone's anisotropic elastic properties were limited in their interpretation by poor precision and resolution of measurement techniques. Here, we report measurements of bone anisotropy using high precision acoustic microscopy. The elastic properties of canine femoral bone specimens, taken from 23 femora, were measured at 10 degrees increments from the long axis of the bone. Half of the bone specimens subsequently were demineralized in EDTA solution, the other half were decollagenized in sodium hypochlorite solution, and the acoustic measurements were repeated. We found the elastic symmetry of osteonal bone deviates significantly from orthotropic theory supporting the hypothesis that the lamellar microstructure forms a "rotated plywood" (Weiner and Traub, FASEB J 6:879-885; 1992). The principal orientation of bone mineral was along the long axis of the bone, while bone collagen appeared to be aligned at a 30 degrees angle to the long axis. The misalignment between the mineral and the collagen suggests that (1) a substantial percentage of the mineral is extrafibrillar, and (2) the alignment of extrafibrillar mineral is governed by external influences, e.g., mechanical stresses.

Acoustics↗
PubMed1993

Scanning acoustic microscope studies of the elastic properties of osteons and osteon lamellae.

Scanning acoustic microscopy (SAM) provides the means for studying the elastic properties of a material at a comparable level of resolution to that obtained by optical microscopy for structural studies. SAM is nondestructive and permits observation of properties in the interior of materials which are optically opaque. Two modes of ultrasonic signals have been used in a Model UH3 Scanning Acoustic Microscope (Olympus Co., Tokyo, Japan) as part of a continuing study of the microstructural properties of bone. The pulse mode, using a single narrow pulse in the range of 30 MHz to 100 MHz, has been used to survey the surface and interior of specimens of human and canine femoral compact cortical bone at resolutions down to approximately 30 microns. To obtain more detailed information at significantly higher resolution, the burst mode, comprised of tens of sinusoids, has been used at frequencies from 200 MHz to 600 MHz. This has provided details of both human and canine single osteons (or haversion systems) and ostenoic lamellae at resolutions down to approximately 1.7 microns, well within the thickness of a lamella as viewed in a specimen cut transverse to the femoral axis.

Animals↗
PubMed2004

Correlation study of scanning acoustic microscope reflection coefficients and image brightness intensities of micrographed osteons.

Scanning acoustic microscopy (SAM) of Haversian bone at high frequency clearly shows differences in the brightness (gray) levels of individual osteons, which were observed to correspond to the differences in reflection coefficients measured with the SAM technique: the darker the bony regions on the SAM micrographs, the lower the SAM reflection coefficient values, and vice versa. We studied the correlation between differences in brightness and the related reflection coefficients of osteons on undecalcified transverse sections of goat tibial diaphysis using a 400-MHz burst mode lens on the Olympus UH3 SAM. Results showed that there was a strong linear correlation between brightness intensities and the corresponding reflection coefficients, with a correlation coefficient r = 0.99 ( P << 0.001) using gray level 210 as cutoff. This result suggested that the image brightness intensity of osteonic bone could be measured retrospectively to provide information on the stiffness of the corresponding bone matrix.

Acoustics↗
PubMed1982

Frequency-dependent angle scattering of ultrasound by liver.

Ultrasonic scattering by one specimen of normal pig liver and two specimens of abnormal human liver has been measured as a function of angle and frequency at each angular position of transmit and receive transducers which were rotated in equal and opposite directions about the axis of a cylindrically shaped tissue sample. Mean data values were determined by averaging points at like frequencies and angular positions in scans made at intervals along the axis of the specimen. Sections of the specimens were obtained throughout its length and stained to emphasize structures containing collagen and connective tissue shown by acoustic microscopy to produce sound speed variations which are known to cause scattering. Using the stained sections as diffraction screens, calculations of scattering were carried out via Fourier transforms on a scale comparable to that in the acoustic studies for comparison with the acoustic data. In all specimens studied, mean acoustic scattering exhibited a general decrease in level with increasing scattering angle. The rate of decrease in acoustic scattering with angle agreed qualitatively with the calculations of average angular falloff of scattering by the stained sections. Lower rates of decrease were observed in tissues with more closely spaced collagen containing structures. Although the data derived from the few substantially different specimens studied is not intended to be representative of all liver, the results show a qualitative correspondence between acoustic scattering and visual appearance which depends on tissue components and their arrangement.

Animals↗
PubMed1997

Regional and temporal changes in the acoustic properties of fracture callus in secondary bone healing.

Controlled fractures were created in the right femora of 17 male Sprague-Dawley rats. The fractured limbs were harvested at 2, 4, 6, and 8 weeks after fracture, fixed, and embedded in polymethylmethacrylate. Midsagittal sections from each animal were evaluated with a scanning acoustic microscope, a device that generates an acoustic impedance map of the scanned material. The impedance of the fracture callus was measured in six regions on each specimen. These regions were chosen in an effort to distinguish between the impedance of the callus formed through intramembranous or endochondral ossification, and we found that the time course of increasing impedance differed for the fracture callus formed through the two pathways. Additionally, we found a significant difference in the mean impedance of the callus at each time period (p < or = 0.0013 for all comparisons), which resulted in an extremely linear relationship (r2 = 0.999) between mean callus impedance and healing time. This experimental model has become a popular choice for the investigation of fracture healing. As such, an accurate determination of the mechanical properties of the fracture callus is often sought. We propose that the implementation of scanning acoustic microscopy in the study of fracture healing may determine the changes in the material properties more accurately than conventional testing methods.

Acoustic Impedance Tests↗
PubMed1996

Influence of tissue preparation on the high-frequency acoustic properties of normal kidney tissue.

The influence of various tissue preparations on the acoustic properties of normal kidney tissue at high frequencies was investigated. Eight surgically excised normal kidney tissue specimens were classified into three groups: (i) fresh, frozen section, (ii) formalin-fixed, frozen section and (iii) formalin-fixed, paraffin section. Scanning acoustic microscopy operating in the frequency range of 100-200 MHz was used to display the two-dimensional distribution of attenuation constant and sound speed. Our results indicate that (i) there is no significant variation in both acoustic parameters between the three tissue groups, (ii) fixation by 10% formalin produces no significant change in the acoustic parameters, (iii) in fat-free tissue regions, the acoustic parameters are independent of preparation method and (iv) frozen sections must be used to assess the acoustic parameters in fat-rich tissues.

Histocytological Preparation Techniques↗
PubMed2000

High-frequency ultrasound characterization of microporous biointegrable polymers in cornea using acoustic parameters.

The current work deals with the use of high-frequency quantitative backscatter acoustic microscopy for the evaluation of the biointegration of microporous polymer implants used as support of artificial cornea. A three-dimensional (3-D) 80 MHz ultrasound microscope (25 microns axial resolution at focus) was used for the imaging and characterization of the progressive biointegration of polymers implanted in rabbit cornea. In-vitro and in-vivo studies were performed. Quantitative assessment of the structural changes in the biomaterial was performed using the spectral analysis of the radio frequency signal and the estimation of acoustic parameters in the 10-65 MHz frequency bandwidth. Correlation of in-vitro ultrasound data with histologic findings has shown that attenuation and backscatter coefficients are sensitive to the changes in the polymer pore content with time. Our results obtained in vivo demonstrated that 3-D 80 MHz echography coupled with quantitative characterization provide a unique tool for the non-invasive and objective follow-up of the implant biointegration and should contribute to clinical management of artificial cornea.

Acoustics↗
PubMed1992

Dynamic non-invasive measurements of arterial diameter and wall thickness.

AIM: Non-invasive measurements of arterial diameter and wall thickness are critical in characterizing the onset and development of vascular disease. A precise dynamic method was proposed and tested for this purpose. DESIGN: A non-invasive method of measuring the variations in diameter and thickness of human arteries throughout the cardiac cycle was developed, using a high-precision ultrasonic echo-tracking system. An adaptive filtering technique was used to suppress artefacts caused by the layered tissue structure of the vessel wall. RESULTS: Based on decorrelation of microstructure noise, this technique improved the detectability of the wall interfaces, which allowed a determination of thickness and diameter. The accuracy and reproducibility of the method were tested by measurements of plastic films with known thicknesses. The discrepancies between standard micrometer and pulse-echo measurement were consistently less than 5 microns for film thicknesses ranging from 220 to 800 microns. The difference between two successive measurements was less than 2 microns. The identity of the measured vascular interfaces was checked in two ways. First, experiments on fixed bovine carotid arteries showed that the identified echogenic interfaces corresponded to the actual anatomical structure, as obtained by acoustic microscopy. Second, the radial artery thickness and diameter were extrapolated to obtain the change in wall volume over one cardiac cycle. The volume was found to be nearly constant, indicating incompressibility. CONCLUSION: This method will make it possible to obtain new information on atherogenesis and other vascular diseases.

Acoustics↗
PubMed2001

Acoustic microscopic analysis of the biological structure of insect wing membranes with emphasis on their waxy surface.

The mechanical performance of natural materials depends on the type, and especially the composition of the molecular constituents. They are almost without exception composite materials, whose characteristics are determined by the characteristics of the individual constituents, their shape, their interaction, and in particular their orientation within the natural material. One of the most impressive natural composites is the insect cuticle. This lightweight building material impresses one with its ability to withstand extremely heavy loading. Even the ultrathin (3-10 microm) membranes of insect wings add greatly to the structural stability of the wings. By means of acoustic microscopy, the present study also shows that the thin covering of wax on the membrane is not an accidental material arrangement. Contrary to that of locust wings, dragonfly wing membranes were found to have a criss-cross fiber-like density gradient within to the waxy layer. This density gradient proved to be mechanically relevant in stabilizing the wings.

Acoustics↗
PubMed2002

High-frequency ultrasound imaging of the intervertebral disc.

In vitro investigation of the detailed structure of the intervertebral disc has been reliant upon microdissection and histological techniques. However, these techniques are laborious and destructive; the latter aspect is important because it precludes repetitive testing of the disc during in vitro experimentation. The aim of the present study was to investigate the potential of using scanning acoustic microscopy (SAM) to image the detailed structure of the intact disc. A mechanically scanned 46-MHz transducer operating in pulse-echo mode was used to obtain images of porcine coccygeal and lumbar discs; scanned regions-of-interest (ROIs) were marked using permanent dye and small surface cuts, respectively. SAM images were compared with corresponding images obtained using conventional histological techniques. The annulus fibrosus was characterised by alternate light and dark bands; the former represent the boundary between adjacent lamellae. Lamellar discontinuities and pathologic abnormalities were observed in specimens. SAM is a novel, nondestructive method for detailed visualisation of the internal structure of the disc in vitro.

Acoustics↗
PubMed2000

Cell property determination from the acoustic microscope generated voltage versus frequency curves.

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

Acoustics↗
PubMed2001

Temperature dependence of ultrasonic enhancement with a site-targeted contrast agent.

Molecular imaging contrast agents specifically detect the biochemical "signatures" of disease before anatomical manifestations are apparent. Sensitive and specific localization of fibrin both in vivo and in vitro has been demonstrated with the use of a ligand-directed liquid perfluorocarbon nanoparticle. Since the acoustic properties of perfluorocarbons are known to vary with temperature, it was hypothesized that temperature could be used to augment the magnitude of enhancement imparted by targeted nanoparticles. Accordingly, the acoustic backscatter of two different substrates, nitrocellulose membrane and human plasma clot, targeted by the nanoparticles was measured at temperatures ranging from 27 degrees to 47 degrees C in 5 degrees C increments. Classic avidin-biotin interactions were utilized to couple biotinylated nanoparticles to avidin-conjugated nitrocellulose membranes. Ultrasonic contrast enhancement of the nitrocellulose membrane at 25 MHz, measured by acoustic microscopy, increased from 2.0+/-0.3 dB at 27 degrees C to 3.7+/-0.4 at 47 degrees C. In a similar experiment, antifibrin nanoparticles bound to human plasma clots also exhibited temperature-dependent ultrasonic signal enhancement ranging from 13.9+/-1.5 dB at 27 degrees C to 18.1+/-1.5 dB at 47 degrees C. The increase in ultrasonic contrast enhancement measured was well described by a simple, acoustic transmission line model with temperature-dependent impedance. These results suggest that temperature-dependent changes in acoustic backscatter may be used to further differentiate tissues targeted with site-specific nanoparticles from surrounding normal soft tissues.

Acoustics↗
PubMed1997

High-frequency ultrasonic detection of thrombi with a targeted contrast system.

Site-targeted acoustic contrast agents used in conjunction with high-frequency intravascular ultrasound have the potential to localize and characterize intravascular pathology. The present study quantifies the utility of a novel, site-targeted ultrasonic contrast agent with high-frequency ultrasound (30 to 50 MHz) and demonstrates the feasibility of the new agent for augmenting detection of targeted pathology with intravascular ultrasonic catheters. High-frequency acoustic microscopy was used to image avidinconjugated nitrocellulose membranes after exposure to a control or biotinylated contrast agent. Increases (p < 0.05) in backscattered power of approximately 66 dB (4-fold) were found for the biotinylated, but not the control contrast agent. Intravascular ultrasonic images (30 MHz nominal center frequency) of plasma clots after exposure to the targeted contrast agent were brighter (p < 0.05) than in controls. These results demonstrate high-frequency acoustic enhancement with a novel targeted contrast agent and may extend the potential diagnostic spectrum of intravascular ultrasound.

Avidin↗
PubMedDate not supplied

Mechanical basis of cell shape: investigations with the scanning acoustic microscope.

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

Acoustics↗
PubMed2001

Assessment of rat articular cartilage maturation using 50-MHz quantitative ultrasonography.

OBJECTIVE: The objective was to assess the relationship between maturation-related structural changes of articular cartilage and variations of acoustic parameters estimated using high frequency ultrasonography. DESIGN: Patellae taken from 48 immature Wistar male rats and divided into six age groups (from five to 11 weeks old) were explored in vitro using 50-MHz scanning acoustic microscopy, then assessed by histology for the analysis of the cartilage cell distribution and fibrillar collagen organization. The variation of cartilage proteoglycan and collagen content with age was evaluated. Thickness measurements performed on both B-scan images and histologic sections were compared. Ultrasonic radiofrequency signals reflected by the cartilage surface and backscattered from its internal matrix were processed to estimate the integrated reflection coefficient (IRC) and apparent integrated backscatter (AIB). RESULTS: One-way ANOVA indicated that acoustic parameters and thickness change significantly (P < 0.05) as the animal matures because of age-related changes in cartilage composition and morphology. A moderate correlation was found between IRC and the animal age. The parameter decreased slightly but significantly over time. However, a good correlation was observed between the rat age and the AIB, which decreased significantly over time. The parameter variation was mostly related to the changes in collagen fiber orientation, and/or to a change in cell size, density and organization. CONCLUSIONS: Current results indicate that acoustic properties of cartilage are affected by maturation-related cartilage changes. This suggests that high frequency ultrasonography may serve as a useful means for the investigation of cartilage matrix structural changes occurring under various clinical circumstances, like those observed during osteoarthritis, and for the evaluation of the efficacy of specific therapeutics.

Aging↗
PubMed2006

Optical and acoustic detection of laser-generated microbubbles in single cells.

Acoustically monitored laser-induced optical breakdown (LIOB) has potential as an important tool to diagnose and treat living cells. Laser-induced intracellular microbubbles are readily detectable using high-frequency ultrasound, and LIOB can be controlled to operate within two distinct regimes. In the nondestructive regime, a single, short-lived bubble can be generated within a cell, without affecting its immediate viability. In the destructive regime, the induced photodisruption quickly can kill a targeted cell. To generate and monitor this range of bioeffects in real time, we have developed a system integrating an ultrafast laser source with optical and acoustic microscopy. Experiments were performed on monolayers of Chinese hamster ovary (CHO) cells. A 793 nm, 100 fs laser pulsed at 3.8 kHz was tightly focused within each cell to produce the photodisruption, and a 50 MHz ultrasonic transducer monitored the resultant bubble via continuous pulse-echo recordings. Photodisruption was also observed using bright field microscopy, and cell viability was assessed following laser exposure with a trypan blue assay. By controlling laser pulse fluence and exposure duration, either nondestructive or destructive LIOB could be produced. The intracellular position of the laser focus was also varied to demonstrate that cell viability was affected by the specific location of material breakdown.

Acoustics↗
PubMed1995

Material and compositional properties of selectively demineralized cortical bone.

Timed immersion in buffered ethylenediamine-tetraacetic acid (EDTA) was used to selectively alter the mineral content at each level in the cortical bone structural hierarchy. The effects on the mechanical behavior were investigated using a combination of experimental techniques which provide collectively a wide range of resolution (5 microns to 3 mm). Optical microscopy and histological analysis demonstrated a heterogeneous structure consisting of a mineralized tissue core surrounded by a layer of demineralized tissue (collagen) whose thickness varied depending on the immersion time. The mechanical behaviors of treated samples with (intact) and without (core) the surrounding demineralized layer were evaluated using three-point flexure. Overall, the intact specimens became significantly less brittle with increased immersion time in buffered-EDTA. For the core specimens, there was a systematic decrease in the elastic flexural properties (E, sigma e, epsilon e). The site-specific properties of the specimens were determined using microhardness testing, scanning acoustic microscopy, and wavelength dispersive analysis. The mineralization and site-specific properties of the mineralized cores were not significantly affected by buffered-EDTA immersion; however, histomorphometric analysis showed a decrease in the mineralized volume fraction via widening of the pre-existing vascular channels. The experimental hierarchy was effective in discerning site-specific property changes and the localized heterogeneities resulting from the buffered-EDTA treatment. Based on the results of this study, buffered-EDTA treatment can be used to facilitate the determination of material and physical properties of intact and demineralized tissues within a single cortical bone sample.

Animals↗
PubMed2002

Effect of articular cartilage proteoglycan depletion on high frequency ultrasound backscatter.

OBJECTIVE: To study the effect of variations of articular cartilage proteoglycans (PG) on high-frequency ultrasound backscatter. DESIGN: The study was performed on patellar cartilages of immature and mature rats (N=36). The variation of PG content was induced by enzyme digestion. Control and treated cartilages were explored in vitro using a 55MHz scanning acoustic microscopy, then assessed by histology for the fibrillar collagen organization analysis. The variations of proteoglycan and collagen content were evaluated. Thickness measurements performed on both B-scan images and histologic sections were compared. Ultrasonic radio-frequency signals reflected by the cartilage surface and backscattered from its internal matrix were processed to estimate the integrated reflection coefficient (IRC) and apparent integrated backscatter (AIB). RESULTS: Although hyaluronidase treatment of immature and mature cartilages removed approximately 50% of the proteoglycans, the echogenicity level of ultrasound images of degraded cartilages was similar to that of controls. IRC and AIB parameters did not significantly vary. Histologic sections of degraded cartilage displayed no change in collagen fiber organization. The thickness mean values measured by ultrasound in PG-depleted groups were significantly higher than in controls, whereas no significant difference in thickness was detected by histological measurement. The increase in cartilage thickness may potentially be explained by a decrease of speed of sound in PG-depleted cartilages that is more likely subsequent to an increase of water content. CONCLUSION: Current results indicate that PG depletion has no significant effect on high frequency ultrasound backscattered from rat patellar cartilage. Ultrasound may provide information about variations of PG content via speed of sound measurement.

Acoustics↗