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 μm 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.

Congenital heart disease results from genetic defects that are manifested at early stages of embryogenesis. The mouse is the preferred animal model for studies of mammalian embryonic development and for an increasing number of human disease models. A number of genes identified in the mouse are critical for normal cardiovascular development, but an understanding of the underlying mechanisms regulating heart development is still incomplete, in part because of the lack of methods to measure hemodynamics in live mouse embryos. We describe the development of a 40-MHz ultrasound scanner, which allows image-guided continuous-wave and pulsed Doppler blood flow measurements in mouse embryos, in utero, at the critical early developmental stages. Doppler waveforms acquired from mouse embryonic umbilical vessels, descending aorta, and cardiac ventricles are presented to demonstrate the utility of the method. By combining image-guided ultrasound Doppler with the many available mouse mutants, this approach should lead to new insights into embryonic cardiovascular structure-function relationships.

PURPOSE: To show that extending Doppler imaging into the high frequency domain could allow detection and characterization of blood flow in small arterioles and capillaries.METHODS: A 40-MHz continuous wave Doppler system and a 60-MHz pulsed-wave Doppler system were constructed, tested, and used to examine the ciliary body region in two normal volunteers.RESULTS: Ciliary body circulation in the region of the great circle of the iris, which is undetectable by conventional 7.5-MHz duplex Doppler, was consistently and reproducibly detectable by high-frequency (40 MHz and 60 MHz) Doppler systems.CONCLUSION: High-frequency Doppler imaging may provide a unique new tool for the characterization and assessment of anterior segment ocular blood flow. (Am J Ophthalmol 1998;126: 597-600, (C) 1998 by Elsevier Science Inc. All rights reserved.).

Extensive genetic information and the recent introduction of transgenic techniques for genetic manipulation have made the mouse the accepted model for normal cardiac development and congenital heart disease. An impediment to progress in this area has been the lack of noninvasive technologies to assess embryonic cardiovascular structure and function. The authors have developed a high frequency (40-50 MHz) ultrasound imaging and Doppler system enabling in vivo analysis of early embryonic cardiovascular development in the mouse. Simultaneous real-time image and Doppler data can be obtained noninvasively allowing, for the first time, measurements to be made of inflow and outflow characteristics of the mouse embryo heart and blood flow in the umbilical circulation. High resolution (measured lateral resolution=60-100 /spl mu/m) images are used to position the sample volume of the Doppler transducers. The 40 MHz CW Doppler system utilizes a pair of air backed LiNbO/sub 3/ transducers mounted on SMB connectors, and has been calibrated with a string phantom from 10 to 300 mm/s, velocities relevant to embryonic cardiovascular blood waveforms. Biphasic inflow waveforms were measured in normal 10 day mouse embryos (approximately equivalent to 4 weeks human) with peak blood velocities close to 200 mm/s. Outflow velocities in the same embryos were close to 70 mm/s while umbilical artery velocities were approximately 20 mm/s. Future studies will compare normal velocity data to measurements of mutant mice with specific defects in heart and placenta.

Basic ultrasound physics and several clinical and experimental observations suggest that high frequency Doppler ultrasound (HFD) operating in the frequency range 20 to 100 MHz holds the promise of a real-time, non-invasive, depth-resolving technique capable of detecting blood flow in the microcirculation with suitable spatial and temporal resolutions. This paper describes a directional, continuous-wave, high frequency Doppler ultrasound (CW HFD) system which we have developed and optimised for use in an investigation to quantify HFD's abilities. The system electronics operates over the frequency range 1 to 200 MHz and has a dynamic range of 100 dB and a noise floor of 10 nV. Using a string phantom and a sensitive 40 MHz transducer we show that HFD can detect and measure velocities of the order of 1 mm/s, the velocity of blood in a capillary. Also, using in vivo measurements we demonstrate that HFD is sensitive to the detection of blood flow in small vessels.

The combined effect of three assumption's relating to refraction, the speed of sound in tissue and the speed of sound in blood on the accuracy of Doppler ultrasound blood velocity measurements has been investigated. A theoretical relationship giving the net velocity measurement error introduced by these three assumptions has been derived using a model in which tissue and blood layers are separated by straight, parallel boundaries. This net error is dependent on the assumed and actual speed of sound in tissue, the assumed speed of sound in blood and the Doppler angle, but is effectively independent of the actual speed of sound in blood, For clinical blood velocity measurements, the net error is estimated to be as much as an 8% overestimation of the actual velocity, higher than previously predicted for any of the factors individually. The relationship also predicts a net velocity measurement error in experimental flow systems and string phantoms which is dependent on the speed of sound in the liquid bath. A water bath at room temperature will give an overestimation of approximately 2%. Experimental investigations using conventional and modified string phantoms and a 5-MHz linear phased array system support these conclusions. The effect of perturbing the layers from their parallel orientation has also been considered theoretically and has provided additional support for the above conclusions. These results may help assure more accurate Doppler velocity measurements in both experimental and clinical settings.

Doppler ultrasound flow measurement techniques are often validated using phantoms that simulate the vasculature, surrounding tissue and blood. Many researchers use rubber tubing to mimic blood vessels because of the realistic acoustic impedance, robust physical properties and wide range of available sizes. However, rubber tubing has a very high acoustic attenuation, which may introduce artefacts into the Doppler measurements. We describe the construction of a wall-less vessel phantom that eliminates the highly attenuating wall and reduces impedance mismatches between the vessel lumen and tissue mimic. An agar-based tissue mimic and a blood mimic are described and their acoustic attenuation coefficients and velocities are characterised. The high attenuation of the latex rubber tubing resulted in pronounced shadowing in B-mode images; however, an image of a wall-less vessel phantom did not show any shadowing. We show that the effects of the highly attenuating latex rubber vessels on Doppler amplitude spectra depend on the vessel diameter and ultrasound beam width. In this study, only small differences were observed in spectra obtained from 0.6 cm inside diameter thin-wall latex, thick-wall latex and wall-less vessel phantoms. However, a computer model predicted that the spectrum obtained from a 0.3-cm inside diameter latex-wall vessel would be significantly different than the spectrum obtained from a wall-less vessel phantom, thus resulting in an overestimation of the average fluid velocity. These results suggest that care must be taken to ensure that the Doppler measurements are not distorted by the highly attenuating wall material. In addition, the results show that a wall-less vessel phantom is preferable when measuring flow in small vessels.