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Assessment of bone structure and acoustic impedance in C3H and BL6 mice using high resolution scanning acoustic microscopy.

Two hundred-MHz time-resolved scanning acoustic microscopy was applied for the investigation of acoustic and structural bone properties of mice from two inbred strains. Transverse sections of femur taken from 5 C57BL/6J@Ico and 5 C3H/HeJ@Ico mice were explored. Both strains had the same bone diameter, but the C3H/HeJ@Ico mice had greater cortical thickness, smaller cancellous diameter, and greater acoustic impedance values than C57BL/6J@Ico mice. The strong differences in the measured acoustic impedances among the two inbred strains indicate that the impedance is a good parameter to detect genetic variations of the skeletal phenotype in small animal models.

Animals↗

An acoustic microscopy technique reveals hidden morphological defenses in Daphnia.

Inducible defenses are common strategies for coping with the selective force of predation in heterogeneous environments. In recent years the conspicuous and often dramatic morphological plasticity of several waterflea species of the genus Daphnia have been found to be inducible defenses activated by chemical cues released by predators. However, the exact defensive mechanisms remained mysterious. Because even some minute morphological alterations proved to be protective against predatory invertebrates, it has been suggested that the visible morphological changes are only the tip of the iceberg of the entire protective mechanisms. Here we applied a method of ultrasonic microscopy with vector contrast at 1.2 GHz to probe hidden morphological defenses. We found that induction with predator kairomones increases the stability of the carapace in two Daphnia species up to 350%. This morphological plasticity provides a major advantage for the induced morphs during predation because predatory invertebrates need to crush or puncture the carapace of their prey to consume them. Our ultrastructural analyses revealed that the internal architecture of the carapace ensures maximal rigidity with minimal material investment. Our results uncover hidden morphological plasticity and suggest a reconsideration of former classification systems in defended and undefended genotypes in Daphnia and possibly in other prey organisms as well.

Animals↗

Sound speed scanning acoustic microscopy for biomedical applications.

Since 1985, we have been developing a scanning acoustic microscope (SAM) system for biomedical use and have been investigating the acoustic properties of various organs and disease states by using this SAM system. In biomedicine, SAM is useful for intraoperative pathological examination, study of low-frequency ultrasonic images, and assessment of biomechanics at a microscopic level. Recently, we have proposed a new concept -- acoustic microscopy -- using a single pulsed wave instead of continuous waves used in conventional SAM systems. In the present study, we compared two systems by measuring the same biological material. The sound speed image obtained by sound speed microscopy corresponded well to that obtained using a conventional SAM system. Lesions with hyaline degeneration showed a lower sound speed when compared with that of normal myocardium. Frequency domain analysis of amplitude and phase by both methods also showed similar characteristics. Although the data acquisition time of one frame was greater than that in conventional SAM, the total time required for calculation was significantly shorter. The SAM system can be applied to intraoperative pathological examination.

Animals↗

The use of acoustic microscopy to study the mechanical properties of glass-ionomer cement.

OBJECTIVES: The aim of the study was to investigate the relationship between microstructure, acoustic and mechanical properties of hardened dental cement samples, prepared with different powder/water ratios. METHODS: Glass-ionomer dental cement samples, prepared with a standard amount of cement powder and different amounts of water have been examined after being hardened. Surface microstructure and ultrasound, longitudinal and shear velocities were obtained with a scanning acoustic microscope. Conditional effective elastic modulus and Poisson's ratio have been calculated using longitudinal and shear sound velocity values. Then on the same samples elastic modulus and microhardness have been determined by standard tests. Additional samples have been used to determine compressive strength. RESULTS: Density; conditionally instantaneous elastic modulus; high-elasticity modulus and compression strength of the samples decrease when large amounts of water were used for their preparation. At the same time porosity and microhardness of the cement matrix increase. Acoustic parameters and parameters of elasticity, calculated on the basis of sound velocity, demonstrated changes, similar to those obtained in standard mechanical tests. SIGNIFICANCE: The established relation between microstructure, acoustic and mechanical parameters demonstrated a high capacity of acoustic microscopy application for non-destructive characterization of dental materials. A particular advantage of the acoustic microscopy is the opportunity to evaluate microstructure and mechanical properties on the same sample.

Compressive Strength↗

Characterization of renal angiomyolipoma by scanning acoustic microscopy.

A scanning acoustic microscope system was used to differentiate renal angiomyolipoma from renal cell carcinoma. The ultrasonic frequency used ranged from 100 to 200 MHz, and the attenuation constant and sound speed were measured on a two-dimensional distribution. The sound speed was significantly lower for lipoma cells than for vessels, smooth muscle fibres, clear cell renal cancer or granular cell renal cancer. The attenuation constant was significantly lower for lipoma cells than for vessels or clear cells. Both acoustic parameters for smooth muscle fibres were significantly lower than for vessels. The heterogeneity of the microacoustic field in renal angiomyolipoma is closely related to the high intensity echo observed on clinical echography. Renal angiomyolipoma and renal cell carcinoma can thus be distinguished by acoustic examination.

Angiomyolipoma↗

Images of arterioles in unfixed tissue obtained by acoustic microscopy.

A scanning acoustic microscope operating at 600 MHz was used to observe arterioles in a thin sheet of collagenous connective tissue dissected from the submucosa of the guinea-pig small intestine. The arterioles were clearly defined in images made using transmitted ultrasound, and the acoustic attenuation (alpha) of the arteriolar wall was estimated to be 120 cm-1. Images made using reflected ultrasound did not show the arterioles clearly.

Animals↗

Observation of ultrasound velocity gradient in fullerene ceramics by acoustic microscopy

A scanning acoustic microscope is used to study the distribution of elastic properties in small samples (O 3 x 2 mm3) of new hard phases of C60. The specimens under investigation were synthesized from pure C60 powder under pressure P = 8 GPa in the temperature range 500-1650 K. The time-of-flight mode was used for bulk sound wave velocity determination in a direction parallel to the cylinder's axis. Longitudinal sound wave velocities greater than 10,000 m/s were found for all specimens treated at temperatures higher than 1000 K. Using the B-scan mode allowed us to observe the velocity gradient in the sample's periphery. The heterogeneous internal structure of the specimen is visualized in the images formed in C- and B-scan modes.

Journal Article↗

In vitro characterization of a novel, tissue-targeted ultrasonic contrast system with acoustic microscopy.

Targeted ultrasonic contrast systems are designed to enhance the reflectivity of selected tissues in vivo [Lanza et al., Circulation 94, 3334 (1996)]. In particular, these agents hold promise for the minimally invasive diagnosis and treatment of a wide array of pathologies, most notably tumors, thromboses, and inflamed tissues. In the present study, acoustic microscopy was used to assess the efficacy of a novel, perfluorocarbon based contrast agent to enhance the inherent acoustic reflectivity of biological and synthetic substrates. Data from these experiments were used to postulate a simple model describing the observed enhancements. Frequency averaged reflectivity (30-55 MHz) was shown to increase 7.0 +/- 1.1 dB for nitrocellulose membranes with targeted contrast. Enhancements of 36.0 +/- 2.3 dB and 8.5 +/- 0.9 dB for plasma and whole blood clots, respectively, were measured between 20 and 35 MHz. A proposed acoustic transmission line model predicted the targeted contrast system would increase the acoustic reflectivity of the nitrocellulose membrane, whole blood clot, and fibrin plasma clot by 2.6, 8.0, and 31.8 dB, respectively. These predictions were in reasonable agreement with the experimental results of this paper. In conclusion, acoustic microscopy provides a rapid and sensitive approach for in vitro chracterization, development, and testing of mathematical models of targeted contrast systems. Given the current demand for targeted contrast systems for medical diagnostic and therapeutic use, the use of acoustic microscopy may provide a useful tool in the development of these agents.

Acoustics↗

Acoustic properties of aortic aneurysm obtained with scanning acoustic microscopy.

In aortic aneurysm tissues, macrophages and their secretion of matrix metalloproteinases (MMPs) are playing important role for tissue degeneration. Some studies have shown that weakening of the mechanical properties of the degenerated tissues may progress the expansion of the aneurysm. However, actual measurement of the mechanical properties has not been investigated at microscopic level. The objective of the present study is to assess the mechanical properties of aortic aneurysm tissues by measuring acoustic properties by scanning acoustic microscopy (SAM). Twenty-one cases of aortic aneurysm including renal and common iliac aneurysm tissues were surgically excised. Each tissue was fixed by 4% formaldehyde and the specimens were treated as (1) picrosirius red staining for normal and polarized light microscopy, (2) CD68 staining for macrophage detection, and (3) no staining for acoustic microscopy. A specially developed SAM system operating in the frequency range of 100-200 MHz, was employed in the measurement. Images of amplitude and phase are obtained in a field of 2x2 mm. The intima was mainly consisted of degenerated collagen without polarization of picrosirius red staining. Macrophages stained by CD68 were observed near the degenerated collagen fibers. The sound speed was 1567 m/s in the intima, 1576 m/s in the media, 1640 m/s in the adventitia, respectively. Infiltration of macrophages showed higher values of attenuation and sound speed than the surrounding tissues. The sound speed of the intima was significantly lower than our previous measurement of atherosclerotic aorta without aneurismal change. As the tissue elasticity is closely correlated with the sound speed, the elasticity of the intima was considered to be lower in aneurysm tissues. This mechanical weakness may contribute to the expansion of the diameter of the aneurysm. Acoustic microscopy provided important data for assessing tissue mechanical properties of abdominal aneurysm.

Acoustics↗

The application of scanning acoustic microscopy in a bone remodeling study.

Scanning acoustic microscopy (SAM) was used in the evaluation of bone remodeling around a cylindrical unicortical defect. SAM is a technique for the nondestructive evaluation of materials, and has only recently been employed as an orthopaedic research tool. The utility of SAM was demonstrated by using it to measure an elastic property known as acoustic impedance. Specifically, the acoustic impedance of bone formed by remodeling around a cylindrical defect was measured. The defects were filled with either a low modulus "void" or rigid inclusion to create various states of stress in the bone in the vicinity of the defect. After six months of implantation of the inclusions in the sheep metatarsal, new bone formation on periosteal and endosteal surfaces about the defect region was observed. These regions of new bone were less stiff and had 18.0 +/- 6.5% lower acoustic impedance than the pre-existing bone in the intracortical region of the metatarsal. There was no difference in the degree of new bone formation about void and rigid inclusions. Both underwent significant adaptational changes in response to the elevated stress about the defect. These changes affected the basic structure of the bone cross-section at the level of the defect and effectively reduced the stress levels about the defect. By using SAM to measure acoustic impedance, it was seen that little internal remodeling occurred in the intracortical region. Hence, the primary mechanism of strain-induced bone remodeling observed in this experimental model was surface remodeling.

Acoustics↗

Identification of hibernating myocardium by acoustic microscopy.

Hibernating myocardium is viable myocardium that recovers after revascularization. The observation of loss of contractile proteins (myofibrils) and accumulation of glycogen in hibernating cardiomyocytes provide the basis for diagnosing hibernating myocardium. In this pilot study, acoustic microscopy was used to identify the cellular structure of normal vs. hibernating myocardium. Sections cut at 5-microm of archival paraffin blocks on glass slides were used for this study. Acoustic microscopy of normal cardiomyocytes showed intracellular linear echoes suggestive of myofibrils, and cardiomyocytes of hibernating myocardium revealed absence of myofibrils and dense intracellular echoes that corresponded to glycogen accumulation on optical microscopy. This modality of visualization allows a definitive diagnosis of hibernating myocardium.

Glycogen↗

Scanning acoustic microscopy study of titanium-ceramic interface of dental restorations.

Failures that occur in titanium-ceramic restorations are of concern in clinical dentistry. The purpose of this study was to nondestructively characterize the internal cracks and nonadherent defects at the titanium-porcelain interface using scanning acoustic microscopy. Titanium samples coated with porcelain without a bonding agent, with sputter coated palladium or chromium as an oxygen diffusion barrier on the titanium, and with the use of a porcelain bonding agent (control group) were compared. The scanning acoustic microscopy analyses were correlated with four-point bending test results. The group that was initially coated with palladium had fewer interfacial defects and a higher load to failure than the control group, and the group that did not contain the bonding agent had a higher void area and a lower load to failure than the control group. The use of chromium produced no differences from the control group. Samples after a four-point bending test were also analyzed by scanning electron microscopy. The scanning electron microscopy was not able to characterize interfacial defects at the fractured titanium-ceramic interface for some of the samples. The validity of nondestructive analysis at the Ti-ceramic interface using scanning acoustic microscopy was demonstrated in this study.

Chromium↗

Evaluation of acoustic properties of the live human smooth-muscle cell using scanning acoustic microscopy.

This study was performed to measure the acoustic propagation speed in live human aortic smooth-muscle cells (HASMC), using scanning acoustic microscopy (SAM) and a novel measurement theory that permits the measurement of the acoustic propagation speed in biological samples of unknown thickness. C-mode and X-Z-mode images of HASMC under three different conditions: growing (G); differential (D); and on hypotonic loading (H), were acquired using 100-MHz, 450-MHz and 600-MHz ultrasound. The images exhibit features related to the cell surface curvature and intracellular structure. The theory supporting the methodology is derived in this article and makes use of the interference fringes within the focusing lens of the high-frequency transducer. The propagation speed in the cells was calculated from the location of the interference fringe on the C-mode images and the fringe shift on the X-Y-mode images with 450-MHz ultrasound. The propagation speed in D (1624 +/- 16 m/s) was significantly higher than those in G (1571 +/- 14 m/s, p < 0.05) and H (1585 +/- 8 m/s, p < 0.05). Scanning acoustic microscope measurements, along with the described theory, are useful for studying the acoustic properties of live cells ex vivo and have applications in both pathophysiology and biomechanics.

Acoustics↗

Detection of Barrett's epithelium by acoustic microscopy.

Barrett's esophagus is associated with increased risk of adenocarcinoma of the gastroesophageal junctional region. The presence of goblet cells (intestinal metaplasia) in columnar cell-lined esophageal mucosa defines Barrett's change. The diagnosis of Barrett's esophagus is based on the presence of intestinal metaplasia in a biopsy from an endoscopically visualized abnormal columnar epithelium. In this pilot study, acoustic microscopy was used to identify the mucosal structure of 10 distal esophageal biopsies. Sections cut at 5 microm of archival paraffin blocks on glass slides were used for this study. Acoustic microscopy permitted the identification of low- and high-power images of epithelial architecture and cellular detail, including Barrett's epithelium. This modality of visualization has the potential to detect lesions such as Barrett's metaplasia, low- and high-grade dysplasia and early carcinoma. If it can be applied to in vivo endoscopy, acoustic microscopy has the potential to increase the accuracy of the diagnosis of Barrett's esophagus, dysplasia and malignancy by providing a method of accurately directing biopsies at endoscopy.

Barrett Esophagus↗

Application of scanning acoustic microscopy for assessing stress distribution in atherosclerotic plaque.

Scanning acoustic microscopy (SAM) was equipped to assess the acoustic properties of normal and atherosclerotic coronary arteries. The SAM image in the atherosclerotic lesion clearly demonstrated that the sound speed was higher than that in the normal intima, and that the variation of elasticity was found within the fibrous cap of the plaque. Young's elastic modulus of each region was calculated and the finite element analysis was applied to derive the stress distribution in these arterial walls. In a case of normal coronary artery, the stress was dominant in the intima and the distribution was rather homogeneous and in a case of atherosclerosis, high stress was concentrated to the relatively soft lesion in the fibrous cap overlying lipid pool. SAM provides information on the physical properties, which cannot be obtained by the optical microscope. The results would help in understanding the pathological features of atherosclerosis.

Acoustics↗

Evaluation of elastic structural change in coronary atherosclerosis using scanning acoustic microscopy.

Coronary atherosclerotic lesions were observed by scanning acoustic microscopy (SAM), a technique which can visualize regions of differing elasticity. Three regions, i.e., dark (dR), intermediate (iR) and bright (bR) demonstrating differences in acoustic reflection intensity and the velocity of surface acoustic waves, were seen in the lesions. Furthermore, lipid-positive areas were found to be dR and iR and fatty crystalline areas were observed only in iR regions by polarizing microscope. These phase transitions of fat affected the acoustic properties in each region. According to SAM images, intimal structural changes were classified into three types, minimum structural change (type I), the overcrowded net-like dR structure (type II) and markedly disturbed structure with a decrease of dR (type III). The medial structure change was also classified into three types paralleling the decrease of dR (types M1, M2 and M3). Intimal type II with a large degree of thickening and intimal type III with medial type M3 were highly prominent in the acute myocardial infarction (AMI) group (P less than 0.01). Therefore these results suggest that the anisotropic elasticity induced by micro-elastic changes in the arterial wall may be associated with functional disturbance of the vascular wall.

Acoustics↗

Ultrasonic tissue characterization of photodamaged skin by scanning acoustic microscopy.

The aim of this study was to ultrasonically characterize photodamaged skin of the elderly at the microscopic level using scanning acoustic microscopy which showed two-dimentional distribution of sound speed in the skin section. We confirmed that the expression level of the elastin gene was increased in the preauricular skin (photodamaged area), compared with postauricular skin (photo-protected area). The expression level of the procollagen gene was also increased in the preauricular skin compared with postauricular skin. The preauricular skin showed higher sound speed in the papillary dermis (Grenz zone). The site of progressive solar elastosis showed a somewhat sound speed velocity than that of the Grenz zone. Immunohistochemical staining showed conserved deposition of collagen in the Grenz zone even in the more photodamaged preauricular skin. These results suggest that fibrosis in the Grenz zone compensates tissue strength with the progress of solar elastosis. The sound speed analysis of skin will provide important information on heterogeneous mechanical changes in the skin during the process of photoaging.

Aged↗

Scanning acoustic microscopy of neoplastic and inflammatory cutaneous tissue specimens.

Acoustic microscopy utilizes high frequency ultrasound to generate microscopic images. The current study was designed to examine representative disorders of the skin by use of a reflective scanning acoustic microscope (R-SAM), and to determine whether the obtainable resolution was sufficient to render a microscopic diagnosis. An Olympus UH3 Scanning Acoustic Microscope was utilized with lenses producing burst wave frequencies at 600 and 800 MHz (600 and 800 million cylces/sec). Cutaneous tissue specimens representing 12 different neoplastic and inflammatory disorders were examined. Acoustic images of unstained sections were compared with conventional light microscopic study of sections stained with hematoxylin-eosin. In most neoplasms examined, it was possible to make a specific diagnosis primarily from low magnification pattern analysis. Although individual cells could be visualized, cytologic atypia was poorly defined. In the inflammatory disorders, a specific diagnosis was possible in all but bullous pemphigoid and lichen planus, because the composition of the inflammatory infiltrate was difficult to determine. The advantages of the R-SAM include the capability of producing an acoustic profile of the tissue and the future possibility of in situ diagnosis.

Humans↗