Daniel H. Turnbull

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.

Beam steering with pulsed two-dimensional transducer arrays

The major problem facing the development of 2-D arrays for imaging is the complexity arising from the large number of elements anticipated in such transducers. The authors have undertaken a theoretical investigation of the focusing and steering properties of pulsed 2-D arrays to characterize the parameters required for medical imaging, such as element size, spacing, and number of elements. Details of the computational methods employed are presented, as well as a discussion of the steered beam properties of wideband 2-D arrays. The effects of apodization and element cross-coupling on the beam properties of a 2-D transducer array are examined. The beam properties of various sparse arrays with elements randomly distributed over the aperture of the transducer are discussed.<<ETX>>

In vivo auditory brain mapping in mice with Mn-enhanced MRI

There are currently no noninvasive imaging methods available for auditory brain mapping in mice, despite the increasing use of genetically engineered mice to study auditory brain development and hearing loss. We developed a manganese-enhanced MRI (MEMRI) method to map regions of accumulated sound-evoked activity in awake, normally behaving mice. To demonstrate its utility for high-resolution (100-μm) brain mapping, we used MEMRI to show the tonotopic organization of the mouse inferior colliculus. To test its efficacy in an experimental setting, we acquired data from mice experiencing unilateral conductive hearing loss at different ages. Larger and persistent changes in auditory brainstem activity resulted when hearing loss occurred before the onset of hearing, showing that early hearing loss biases the response toward the functional ear. Thus, MEMRI provides a sensitive and effective method for mapping the mouse auditory brainstem and has great potential for a range of functional neuroimaging studies in normal and mutant mice.

Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice

To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles express less than 10% of truncated protein in M-bands of otherwise normal sarcomeres. Homozygous mice bearing mutated MyBP-C alleles are viable but exhibit neonatal onset of a progressive dilated cardiomyopathy with prominent histopathology of myocyte hypertrophy, myofibrillar disarray, fibrosis, and dystrophic calcification. Echocardiography of homozygous mutant mice showed left ventricular dilation and reduced contractile function at birth; myocardial hypertrophy increased as the animals matured. Left-ventricular pressure-volume analyses in adult homozygous mutant mice demonstrated depressed systolic contractility with diastolic dysfunction. These data revise our understanding of the role that MyBP-C plays in myofibrillogenesis during cardiac development and indicate the importance of this protein for long-term sarcomere function and normal cardiac morphology. We also propose that mice bearing homozygous familial hypertrophic cardiomyopathy-causing mutations may provide useful tools for predicting the severity of disease that these mutations will cause in humans.

Detection of Alzheimer's amyloid in transgenic mice using magnetic resonance microimaging.

The presence of amyloid‐β (Aβ) plaques in the brain is a hallmark pathological feature of Alzheimers disease (AD). Transgenic mice overexpressing mutant amyloid precursor protein (APP), or both mutant APP and presenilin‐1 (APP/PS1), develop Aβ plaques similar to those in AD patients, and have been proposed as animal models in which to test experimental therapeutic approaches for the clearance of Aβ. However, at present there is no in vivo whole‐brain imaging method to detect Aβ plaques in mice or men. A novel method is presented to detect Aβ plaques in the brains of transgenic mice by magnetic resonance microimaging (μMRI). This method uses Aβ1‐40 peptide, known for its high binding affinity to Aβ, magnetically labeled with either gadolinium (Gd) or monocrystalline iron oxide nanoparticles (MION). Intraarterial injection of magnetically labeled Aβ1‐40, with mannitol to transiently open the blood–brain barrier (BBB), enabled the detection of many Aβ plaques. Furthermore, the numerical density of Aβ plaques detected by μMRI and by immunohistochemistry showed excellent correlation. This approach provides an in vivo method to detect Aβ in AD transgenic mice, and suggests that diagnostic MRI methods to detect Aβ in AD patients may ultimately be feasible. Magn Reson Med 50:293–302, 2003.

Dynamic, contrast-enhanced perfusion MRI in mouse gliomas: Correlation with histopathology

The aim of this study was to develop an MRI protocol to evaluate the growth and vascularity of implanted GL261 mouse gliomas on a 7T microimaging system. Both conventional T1‐ and T2‐weighted imaging and dynamic, contrast‐enhanced T2*‐weighted imaging were performed on 34 mice at different stages of tumor development. MRI measurements of relative cerebral blood volume (rCBV) were compared to histological assessments of microvascular density (MVD). Enhancement on postcontrast T1‐weighted images was compared to histological assessments of Evans blue extravasation. Conventional T2‐weighted and postcontrast T1‐weighted images demonstrated tumor growth characteristics consistent with previous descriptions of GL261 glioma. Furthermore, measurements of rCBV from MRI data were in good agreement with histological measurements of MVD from the same tumors. Postcontrast enhancement on T1‐weighted images was observed at all stages of GL261 glioma progression, even before evidence of angiogenesis, indicating that the mechanism of conventional contrast enhancement in MRI does not require neovascularization. These results provide quantitative support for MRI approaches currently used to assess human brain tumors, and form the basis for future studies of angiogenesis in genetically engineered mouse brain tumor models. Magn Reson Med 49:848–855, 2003.

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.

A method for rapid gain-of- function studies in the mouse embryonic nervous system

We used ultrasound image-guided injections of high-titer retroviral vectorsto obtain widespread introduction of genes into the mouse nervous system in utero as early as embryonic day 8.5 (E8.5). The vectors used includedinternal promoters that substantially improved proviral gene expression inthe ventricular zone of the brain. To demonstrate the utility of this system,we extended our previous work in vitro by infecting the telencephalon in vivo as early as E8.5 with a virus expressing Sonic Hedgehog. Infectedembryos showed gross morphological brain defects, as well as ectopic expressionof ventral telencephalic markers characteristic of either the medial or lateralganglionic eminences.

Specification of Mouse Telencephalic and Mid-Hindbrain Progenitors Following Heterotopic Ultrasound-Guided Embryonic Transplantation

We have demonstrated the utility of ultrasound backscatter microscopy for targeted intraparenchymal injections into embryonic day (E) 13.5 mouse embryos. This system has been used to test the degree of commitment present in neural progenitors from the embryonic ventral telencephalon and mid-hindbrain region. Many E13.5 ventral telencephalic progenitors were observed to integrate and adopt local phenotypes following heterotopic transplantation into telencephalic or mid-hindbrain targets, whereas mid-hindbrain cells of the same stage were unable to integrate and change fate in the telencephalon. In contrast, many mid-hindbrain cells from an earlier developmental stage (E10.5) were capable of integrating and adopting a forebrain phenotype after grafting into the telencephalon, suggesting that mouse mid-hindbrain progenitors become restricted in their developmental potential between E10.5 and E13.5.

Design and fabrication of a 40-MHz annular array transducer

This paper investigates the feasibility of fabricating a five-ring, focused annular array transducer operating at 40 MHz. The active piezoelectric material of the transducer was a 9-/spl mu/m thick polyvinylidene fluoride (PVDF) film. One side of the PVDF was metallized with gold and forms the ground plane of the transducer. The array pattern of the transducer and electrical traces to each annulus were formed on a copper-clad polyimide film. The PVDF and polyimide were bonded with a thin layer of epoxy, pressed into a spherically curved shape, then back filled with epoxy. A five-ring transducer with equal area elements and 100 /spl mu/m kerfs between annuli was fabricated and tested. The transducer had a total aperture of 6 mm and a geometric focus of 12 mm. The pulse/echo response from a quartz plate located at the geometric focus, two-way insertion loss (IL), complex impedance, electrical crosstalk, and lateral beamwidth all were measured for each annulus. The complex impedance data from each element were used to perform electrical matching, and the measurements were repeated. After impedance matching, f/sub c/ /spl sim/ 36 MHz and -6-dB bandwidths ranged from 31 to 39%. The ILs for the matched annuli ranged from -28 to -38 dB.