Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos.

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

Model for the ultrasound reflection from micro-beads and cells distributed in layers on a uniform surface.

A model predicting the reflection of ultrasound from multiple layers of small scattering spheres is developed. Predictions of the reflection coefficient, which takes into account the interferences between the different sphere layers, are compared to measurements performed in the 10-80 MHz and 15-35 MHz frequency range with layers of glass beads and spherical acute myeloid leukemia (AML) cells, respectively. For both types of scatterers, the reflection coefficient increases as a function of their density on the surface for less than three superimposed layers, at which point it saturates at 0.38 for glass beads and 0.02 for AML cells. Above three layers, oscillations of the reflection coefficient due to constructive or destructive interference between layers are observed experimentally and are accurately predicted by the model. The use of such a model could lead to a better understanding of the structures observed in layered tissue images.

Measurement of the ultrasonic properties of human coronary arteries in vitro with a 50-MHz acoustic microscope

Ultrasonic attenuation coefficient, wave propagation speed and integrated backscatter coefficient (IBC) of human coronary arteries were measured in vitro over the -6 dB frequency bandwidth (36 to 67 MHz) of a focused ultrasound transducer (50 MHz, focal distance 5.7 mm, f/number 1.7). Corrections were made for diffraction effects. Normal and diseased coronary artery sub-samples (N = 38) were obtained from 10 individuals at autopsy. The measured mean ± SD of the wave speed (average over the entire vessel wall thickness) was 1581.04 ± 53.88 m/s. At 50 MHz, the average attenuation coefficient was 4.99 ± 1.33 dB/mm with a frequency dependence term of 1.55 ± 0.18 determined over the 36- to 67-MHz frequency range. The IBC values were: 17.42 ± 13.02 (sr.m)-1 for thickened intima, 11.35 ± 6.54 (sr.m)-1 for fibrotic intima, 39.93 ± 50.95 (sr.m)-1 for plaque, 4.26 ± 2.34 (sr.m)-1 for foam cells, 5.12 ± 5.85 (sr.m)-1 for media and 21.26 ± 31.77 (sr.m)-1 for adventitia layers. The IBC results indicate the possibility for ultrasound characterization of human coronary artery wall tissue layer, including the situations of diseased arteries with the presence of thickened intima, fibrotic intima and plaque. The mean IBC normalized with respect to the mean IBC of the media layer seems promising for use as a parameter to differentiate a plaque or a thickened intima from a fibrotic intima

Advances in ultrasound biomicroscopy.

The visualisation of living tissues at microscopic resolution is attracting attention in several fields. In medicine, the goals are to image healthy and diseased tissue with the aim of providing information previously only available from biopsy samples. In basic biology, the goal may be to image biological models of human disease or to conduct longitudinal studies of small-animal development. High-frequency ultrasonic imaging (ultrasound biomicroscopy) offers unique advantages for these applications. In this paper, the development of ultrasound biomicroscopy is reviewed. Aspects of transducer development, systems design and tissue properties are presented to provide a foundation for medical and biological applications. The majority of applications appear to be developing in the 40-60-MHz frequency range, where resolution on the order of 50 microm can be achieved. Doppler processing in this frequency range is beginning to emerge and some examples of current achievements will be highlighted. The current state of the art is reviewed for medical applications in ophthalmology, intravascular ultrasound, dermatology, and cartilage imaging. Ultrasound biomicroscopic studies of mouse embryonic development and tumour biology are presented. Speculation on the continuing evolution of ultrasound biomicroscopy will be discussed.

High-frequency color flow imaging of the microcirculation.

The extension of ultrasound (US) color flow imaging (CFI) techniques to high frequencies (> 20 MHz) has the potential to provide valuable noninvasive tools for scientific and clinical investigations of blood flow in the microcirculation. We describe the development of a slow-scan CFI system operating in the 20-100-MHz range that has been optimized to image the microcirculation. The apparatus has incorporated elements of a previously reported pulsed-wave Doppler system and is capable of operating in either CFI or pulsed-wave mode. The performance of the CFI system was evaluated at a center frequency of 50 MHz using two PVDF transducers with -6-dB beam widths of 43 and 60 microm. The -6 dB-axial resolutions were estimated to be 66 and 72 microm, respectively. In vivo validation experiments conducted using the murine ear model demonstrated the detection of flow in vessels down to 15-20 microm in diameter with flow velocities on the order of mm per s. Further experiments examining experimental murine tumors confirmed the successful detection of flow in the tumor microcirculation.

Ultrasound for the visualization and quantification of tumor microcirculation.

Advances in ultrasound based methods for the non-invasive assessment of the tumor microcirculation are described. Two new ultrasound approaches are highlighted. The first method relies on commercially available ultrasound contrast agents in combination with specialized nonlinear imaging sequences. Nonlinear scattering by microbubble contrast agents provides a unique intravascular signature that can be distinguished from the echoes caused by surrounding tissues. Through destruction-reperfusion experiments it is possible to use microbubble contrast agents as a tracer revealing the kinetics of tumor blood flow. The second ultrasound method for examining the microcirculation involves the use of much higher frequencies. At frequencies on the order of 50 MHz, Doppler processing allows the direct assessment of flow dynamics in realtime in arterioles as small as 15 microm. Three dimensional Doppler maps of flow patterning are presented. The strengths and weaknesses of these new methods are discussed and the potential for their use in preclinical animal drug studies, clinical drug trials, and prognostic studies is described.

Ultrasound biomicroscopy. High-frequency ultrasound imaging of the eye at microscopic resolution.

UBM presents us with a new method of imaging the anterior segment of the eye at high resolution. Its strengths lie in its ability to produce cross-sections of the living eye at microscopic resolution without violating the integrity of the globe. UBM, although lacking the resolution of optical microscopy, gives us images in living eyes without affecting the internal relationships of the structures imaged. There are many other applications of this new imaging method. Examples of other uses include imaging adnexal pathology, assessing corneal changes with refractive surgery, the assessment of trauma, and determination of intraocular lens position.

Ultrasound biomicroscopic imaging of the anterior aspect of peripheral choroidal melanomas.

PURPOSE: To correlate ultrasound biomicroscopic features of the anterior aspect of peripheral choroidal melanoma with respect to histopathology. METHODS: We examined 17 eyes of 17 patients who had clinically diagnosed peripheral choroidal melanomas that approached the ora serrata or extended into the ciliary body and who had been assessed with ultrasound biomicroscopy before enucleation. Comparisons were made between anterior tumor margins imaged by ultrasound biomicroscopy and histopathologic specimens. Anatomic features noted on ultrasound biomicroscopy before enucleation were correlated with enucleation specimens, including supraciliary effusion, rotation of the ciliary body, angle involvement, and internal reflectivity patterns. Anterior tumor margin position was determined with reference to the scleral spur. RESULTS: Mean distances from the anterior tumor margin to the scleral spur were 1.47 mm on ultrasound biomicroscopy and 1.65 mm on pathologic examination. This difference was not statistically significant (P = .325). Tumor features evident on ultrasound biomicroscopy were also seen on pathologic examination: supraciliary choroidal effusions in seven of seven, ciliary body rotation in seven of eight, and angle involvement in seven of eight. All tumors were mixed-cell melanomas, and 12 of 17 (70%) demonstrated homogeneous ultrasound biomicroscopic internal reflectivity. Irregular internal reflectivity was seen in five of 17 tumors (29%) and was related to prominent internal vascularity on pathology in three of five. CONCLUSIONS: Ultrasound biomicroscopy is an accurate imaging technique for the in vivo assessment of anterior tumor margins of peripheral choroidal melanomas and can provide detailed imaging of the tumor's interface with the ciliary body.

Supraciliary effusions and ciliary body thickening after scleral buckling procedures.

PURPOSE: The purpose of the study was to use ultrasound biomicroscopy to identify and quantity changes in anterior segment parameters after scleral buckling procedures. METHODS: Ultrasound biomicroscopy was used to examine 15 patients with retinal detachment within 1 week before and after surgery. Quantitative measurements were performed of anterior chamber depth, supraciliary effusion depth, ciliary body thickness, and angle opening. RESULTS: Supraciliary fluid was present after surgery in 12 patients (80%). Average supraciliary fluid depth was 0.16 +/- 0.13 mm. Ciliary body thickness measurements at a point 2-mm posterior to the scleral spur increased after surgery in all patients an average of 0.15 +/- 0.10 mm. There was a strong correlation between ciliary fluid levels and change in ciliary body thickness (r = 0.742, P < 0.01). Anterior chamber depth decreased after surgery in 14 patients (93%). A decrease of angle opening of greater than 5 degrees was noted in 11 patients (73%). In all of these 11 patients, the ciliary body and iris root were considered to be rotated anteriorly. Six (55%) of 11 of these patients showed anterior bowing of the iris, indicating pupillary block. Complete angle closure occurred over one to three quadrants in three patients, but none of these patients had complete angle closure or glaucoma. CONCLUSIONS: Supraciliary effusions and ciliary body thickening are common after scleral buckling procedures and can produce conditions conducive to angle closure. Angle narrowing occurs through a combination of direct anterior iris rotation and induced pupillary block.

High frequency 40-MHz ultrasound. A possible noninvasive method for the assessment of the boundary of basal cell carcinomas.

BACKGROUND: Ultrasound imaging systems operating close to 20 MHz in frequency have been used to image skin tumors. Ultrasound imaging at 20 MHz has been used to determine the boundaries of basal cell carcinomas (BCCs). An inherent shortcoming of imaging systems operating at these frequencies is their limited resolution. OBJECTIVE: We investigated whether 40-MHz ultrasound imaging could provide higher resolution compared with the lower frequency systems and thus be a superior, noninvasive method of assessing the boundaries of BCCs. METHODS: Nine BCCs from six individuals were examined clinically and ultrasonographically, and then biopsied to confirm diagnosis. The depth of BCCs measured on histological sections was compared with the corresponding value obtained using ultrasound. For this study we required a nonsurgical, nondestructive means of treating BCCs that would allow repeated ultrasound imaging, and therefore topical 5-flurouracil (5-FU) was chosen. Following 5-FU therapy a biopsy was obtained from the site of the treated BCC after ultrasound imaging had been performed. Clinical, ultrasonic and histopathologic evaluation of each BCC was carried out independently by different individuals. At the end of the study all the BCC sites were treated surgically be electrodesiccation and curettage or completely excised. RESULTS: High resolution ultrasound images of BCCs were obtained with agreement between histology and ultrasound findings in all none lesions prior to therapy and in eight of none lesions posttherapy. There was a significant correlation between the depth of BCCs measured histologically and using ultrasound (P = 0.0004, r = 0.92). CONCLUSIONS: This study suggests that 40-MHz ultrasound may provide an estimate of the boundary of a BCC in vivo. High frequency 40-MHz ultrasound imaging may be an adjunct to clinical and histologic evaluation but does not replace the need to obtain tissue for microscopic examination.

Accommodation and iridotomy in the pigment dispersion syndrome.

BACKGROUND AND OBJECTIVE: Iris concavity has been noted in pigment dispersion syndrome, and could have a role in producing iris-zonule contact. Iris concavity is most likely caused by a relative increase in anterior chamber pressure. The method by which this occurs remains speculative. The authors used ultrasound biomicroscopy to examine the role of accommodation in producing iris concavity and to document changes that occur following iridotomy. PATIENTS AND METHODS: Thirteen patients with clinically diagnosed pigmentary dispersion and pigmentary glaucoma underwent accommodation studies while being continuously imaged with ultrasound biomicroscopy. Anterior chamber depths were measured and iris configuration noted on distance and near fixation. These studies were repeated in 6 patients following laser iridotomy. RESULTS: All patients showed a decrease in anterior chamber depth with accommodation. Ten patients had a planar iris configuration on distance fixation and 3 concave. Eleven of 13 patients showed increased concavity of the iris on near fixation as compared with distance fixation. Following iridotomy in 6 patients, the iris showed a planar configuration that remained unchanged on near fixation. CONCLUSION: Accommodation increases iris concavity in some patients with pigment dispersion syndrome. The most likely explanation is an accommodation-induced relative increase in anterior chamber pressure secondary to anterior movement of the lens surface. Iridotomy prevents change in the iris profile with accommodation.

Diagnosis of traumatic cyclodialysis by ultrasound biomicroscopy.

BACKGROUND AND OBJECTIVE: To evaluate the ability of high-frequency ultrasound biomicroscopy to diagnose traumatic cyclodialyses not evident on clinical examination. PATIENTS AND METHODS: Six eyes to six patients with posttraumatic hypotony and/or shallow anterior chamber and suspected cyclodialysis clefts were examined with slit-lamp biomicroscopy, gonioscopy, B-scan ultrasonography, and ultrasound biomicroscopy. Ultrasound biomicroscopy provided high resolution of cross-sectional images of the anterior chamber angle, posterior chamber, and anterior uveal tissue. RESULTS: Ultrasound biomicroscopy confirmed the disinsertion of the ciliary body from the scleral spur and associated ciliary body detachment in all eyes. Gonioscopy failed to demonstrate a cyclodialysis cleft in five eyes because of hyphema (two eyes) and abnormal iris architecture (related to trauma) precluding visualization of the angle recess (three eyes). Using information from ultrasound biomicroscopy imagining, one patient underwent a ciliary body reattachment procedure and repair of the cyclodialysis cleft. CONCLUSION: Ultrasound biomicroscopy is a noninvasive method that can accurately diagnose the presence of traumatic cyclodialyses and can aid in surgical management. It is particularly useful in the presence of hazy media, hypotony, and/or abnormal anterior segment anatomy.

Ultrasound backscatter microscope analysis of mouse melanoma progression.

The incidence and mortality rate of cutaneous melanoma continue to increase throughout the world, making the study of melanoma biology an important area of current research. While recent breakthroughs in transgenic mouse technology have led to promising mouse skin models of melanoma, there is presently no technique available for quantitatively studying subsurface melanoma progression, in vivo. We demonstrate the first application of an imaging method called ultrasound backscatter microscopy (UBM) for imaging early murine melanomas with spatial resolution of 30 microns axial and 60 microns lateral. Murine B16 F10 melanomas have been imaged from their earliest detection, over several days, until they are 2 to 5 mm in diameter. Melanoma dimensions measured by UBM were found to be in excellent agreement with those determined histopathologically on the excised tumours. The relative rms errors in UBM-determined melanoma height and width were found to be 8.7% and 4.2%, respectively. The mean rate of increase in tumour height of early murine melanoma was found to be 0.37 +/- 0.06 mm/day. Computer-generated volumetric renderings of melanomas have been produced from three-dimensional image data, allowing quantitative comparisons of tumour volumes to be made. Using a priori assumptions of ellipsoid tumour shape, the relative error in UBM-determined volume was shown to be less than 17%. These results should be of considerable interest to investigators studying melanoma biology using mouse skin models, and have implications in the use of high frequency ultrasound imaging for the clinical assessment of cutaneous melanoma.

Does high-frequency (40-60 MHz) ultrasound imaging play a role in the clinical management of cutaneous melanoma?

The assessment of cutaneous melanoma in the clinical setting is often difficult, and important features such as depth and width remain unknown until the pathology report is received. Access to prognostic features such as vertical height before excisional biopsy would offer a basis for guidance in defining surgical margins and early planning of treatment options. Recently developed high-frequency ultrasound imaging in the 40-to 60-MHz range is a noninvasive method that provides in vivo information about cutaneous lesions. Imaging at these frequencies provides high-resolution data within the range of the epidermis and dermis (3-4 mm in depth). Ten cutaneous melanomas and seven pigmented lesions were assessed in this fashion. Vertical height was documented and compared to histopathological findings. High-frequency ultrasound imaging determination of vertical height correlated well with the standard measurement of Breslow's thickness on histological sections only in midrange (1.0-3.0 mm) lesions. Inflammatory cells at the base of three melanomas provoked an overestimation of the depth measurement with ultrasonography. Thick keratin layers such as those found on the feet acted as a virtual block to the high-frequency scanner. The application of this new advance in noninvasive imaging technology to the clinical assessment of cutaneous melanoma provides interesting in vivo data but in its present state does not replace the need for the biopsy of pigmented lesions and histopathological diagnosis.

An ultrasound biomicroscopic dark-room provocative test.

Ultrasound biomicroscopy can image the relationship of angle structures under any lighting conditions. Eight patients with narrow angles were examined in the light and in the dark. All of the eyes showed iris thickening and shortening, increased anterior convexity of the iris, and varying degrees of angle narrowing in the dark. In one eye, the angle was completely closed. Iridotomy in this patient flattened the iris profile and opened the angle. The degree of angle opening observed after the iridotomy did not change depending on the lighting conditions. Ultrasound biomicroscopy allows imaging of dynamic changes in anterior ocular structures as they occur and provides information regarding angle occludability in the dark.

Ultrasound backscatter microscope analysis of early mouse embryonic brain development.

The history of developmental and genetic analysis in the mouse has made it the model of choice for studying mammalian embryogenesis. Presently lacking is a simple technique for efficiently analyzing early mouse mutant phenotypes in utero. We demonstrate application of a real-time imaging method called ultrasound backscatter microscopy for visualizing mouse early embryonic neural tubes and hearts. This method was used to study live embryos in utero between 9.5 and 11.5 days of embryogenesis, with a spatial resolution close to 50 microns. Ultrasound backscatter microscope images of cultured embryos made it possible to visualize the heart chambers. This noninvasive imaging method was also used for analyzing a neural tube defect. The midhindbrain deletion associated with a null mutation of the Wnt-1 protooncogene was easily recognizable on ultrasound backscatter microscope images of 10.5- and 11.5-day embryos. Computer-generated volumetric renderings of the neural tube cavities were made from three-dimensional image data. This allowed a much clearer definition of the Wnt-1 mutant phenotype. These imaging techniques should be of considerable use in studying mouse development in utero.

A 40-100 MHz B-scan ultrasound backscatter microscope for skin imaging.

There is a growing interest in high resolution, subsurface imaging of cutaneous tissues using higher frequency ultrasound, and several commercial systems have been developed recently which operate at 20 MHz. Some of the possible applications of higher frequency skin imaging include tumour staging, boundary definition, and studies of the response of tumours to therapy, investigations of inflammatory skin conditions such as psoriasis and eczema, and basic studies of skin aging, sun damage and the effects of irritants. Investigation of these areas is quite new, and the role of ultrasound skin imaging is continuing to evolve. Lateral resolution in the 20 MHz imaging systems ranges from 200 to 300 microns, which limits imaging applications to cutaneous structures which are relatively large in size. In this paper, a real-time ultrasound backscatter microscope (UBM) for skin imaging is described which operates in the 40-100 MHz range, providing axial resolution between 17 and 30 microns and lateral resolution between 33 and 94 microns. This improvement in resolution over current skin ultrasound systems should prove useful in determining the margins of small skin lesions, and in obtaining more precise, in vivo skin thickness measurements to characterize nonmalignant skin disease. Example images of normal skin, seborrhoeic keratosis and malignant melanoma illustrate the imaging potential of this system.

Malignant glaucoma. Clinical and ultrasound biomicroscopic features.

PURPOSE: To determine conditions associated with malignant glaucoma and to assess anterior segment structures in malignant glaucoma using ultrasound biomicroscopy. METHODS: Over a 3-year period, 14 eyes from 14 patients with malignant glaucoma were evaluated. RESULTS: Of the 14 eyes, 7 received medical therapy alone and 7 required both medical and surgical intervention. Predisposing factors included trabeculectomy in 12 eyes, chronic angle-closure glaucoma in 10, sudden ocular decompression in 2 (1 from suture lysis and 1 from corneal perforation), pseudoexfoliation in 4, and cessation of aqueous suppressants and cycloplegics in 2. Ultrasound biomicroscopy of two eyes showed a shallow supraciliary fluid level, anterior rotation of ciliary processes, and obstruction of the trabeculectomy osteum. CONCLUSIONS: Malignant glaucoma is associated with glaucoma surgery, chronic angle-closure glaucoma, pseudoexfoliation, sudden ocular decompression, and cessation of aqueous suppressants. On ultrasound biomicroscopy, a low supraciliary fluid level was found in two patients.

Ultrasound biomicroscopic analysis of haptic position in late-onset, recurrent hyphema after posterior chamber lens implantation.

Late-onset, recurrent hyphema is an uncommon complication of posterior chamber intraocular lens implantation. Pathological studies and indirect clinical evidence, including iris transillumination defects and iris tucking, have implicated haptic-iris contact. The technique of high-resolution ultrasound biomicroscopy developed in our laboratories allows imaging of intraocular lens haptics and their relationship to surrounding structures. Examination of two cases with late-onset, recurrent hyphema revealed that the margins of the superior haptics were sulcus fixated and indenting the peripheral iris. Blood could be traced beneath the peripheral iris in one case. Our findings indicate that haptic-iris contact was the source of bleeding. Capsular fixation should make this complication less likely.