Original contribution
A new ultrasound instrument for in vivo microimaging of mice

https://doi.org/10.1016/S0301-5629(02)00567-7Get rights and content

Abstract

We report here on the design and evaluation of the first high-frequency ultrasound (US) imaging system specifically designed for microimaging of the mouse. High-frequency US or US biomicroscopy (UBM) has the advantage of low cost, rapid imaging speed, portability and high resolution. In combination with the ability to provide functional information on blood flow, UBM provides a powerful method for the investigation of development and disease models. The new UBM imaging system is demonstrated for mouse development from day 5.5 of embryogenesis through to the adult mouse. At a frequency of 40 MHz, the resolution voxel of the new mouse scanner measures 57 μm × 57 μm × 40 μm. Duplex Doppler provides blood velocity sensitivity to the mm per s range, consistent with flow in the microcirculation, and can readily detect blood flow in the embryonic mouse heart, aorta, liver and placenta. Noninvasive UBM assessment of development shows striking similarity to invasive atlases of mouse anatomy. The most detailed noninvasive in vivo images of mouse embryonic development achieved using any imaging method are presented. (E-mail: [email protected])

Introduction

As human and mouse genetic sequencing projects near completion, the next and greater challenge will be to define the roles of tens of thousands of genes in the context of complex organisms Burley et al 1999, Clark 1999, Bentley 2000. The mouse has emerged as one of the models of choice (Marshall 2000) for such studies. Not only do we share over 90% of our genes with the mouse, but this mammal is prolific and inexpensive to house. Over the past several decades, researchers have studied naturally occurring mutations and have learned to manipulate the mouse genome in a targeted and predictable fashion using transgenes and knockouts (Battey et al. 1999). In addition, large-scale random chemical mutagenesis studies (Hrabe de Angelis et al. 2000) are being conducted to identify novel genes involved in human diseases. Thus, there is a significant need for rapid, high-throughput tests to screen for critical geneotype-phenotype relationships. New screening methodologies will include rapid methods for behavioral analysis, automated physiological screens and systems for biochemical profiling of blood and urine. A variety of optical and nonoptical imaging techniques, including US biomicroscopy (UBM), magnetic resonance (MR) microscopy, computed tomographic (CT) microscopy and positron emmision tomography (PET) will be added to this list because they are likely the only means of acquiring anatomic and spatially mapped functional information about living animals. Although UBM does not provide molecular specificity, it has the advantage of low cost, rapid imaging speed, portability and high resolution.

Several groups have actively conducted mouse imaging research using diagnostic US instrumentation operating in the 7.5- to 12-MHz frequency range where resolution is on the order of 300 to 500 μm Fentzke et al 1997, Fentzke et al 1998, Mor-Avi et al 1999, Scherrer-Crosbie et al 1998, Scherrer-Crosbie et al 1999. Investigations of mouse models of myocardial infarction have been performed using contrast agents (Scherrer-Crosbie et al. 1999) and studies of mouse right ventricular function have been undertaken using transesophageal imaging (Scherrer-Crosbie et al. 1998). It has also been possible to examine transgenic models of cardiac hypertrophy with some success (Fentzke et al. 1998). Applications of conventional phased-array US in the mouse will undoubtedly continue to improve as array transducers and signal processing in these systems are moved to higher frequencies. Although conventional US can, in some cases, provide useful information, resolution is marginal and useful observations of neonates and embryonic development are all but impossible. Scaling diagnostic US instruments for applications in the mouse requires several important modifications. In terms of linear dimensions, the most obvious requirement is the need for a scaling factor of approximately 10. For example, the mouse heart measures about 10 to 12 mm on the long axis, whereas the human heart measures approximately 12 to 15 cm. The ratio of the dimensions of other mouse organs to human organs is similar. Optimal imaging of the mouse, therefore, requires an approximately 10-fold improvement in resolution if the same level of structural detail within organs is to be observed. Because resolution scales directly with frequency, the required level of resolution can be achieved by employing much higher US frequencies in the 20 to 60 MHz range. Specialized scanning systems (US biomicroscopes) operating in this frequency range have recently become available for applications in clinical imaging of the eye and skin and in intravascular imaging Foster et al 2000c, Pavlin and Foster 1995, Silverman et al 1997. UBM has also been tested as a tool for mouse imaging Foster et al 2000c, Turnbull 1999, Aristizabal et al 1998, Srinivasan et al 1998, Turnbull et al 1995, Turnbull et al 1996. Turnbull et al. (1995) first reported the use of UBM to observe mutant phenotyping in the mouse embryo. Since that time, improvements in the technology have permitted numerous other investigations in the mouse to be performed (Foster et al. 2000c) using prototype scanners built in our laboratory. In particular, the development of high-frequency continuous-wave (CW) (Christopher et al. 1996), pulsed-wave (PW) (Christopher et al. 1997), and color Doppler Kruse et al 1998, Goertz et al 2000 have enabled the measurement and characterization of the microcirculation to be achieved. Another important innovation has been the development of methodologies for the guided injection of genetic material to specific sites in the developing mouse embryo (Liu et al. 1998).

The technical advances of the past few years have now been consolidated into a new US mouse imaging system that has recently become commercially available (VisualSonics VS40, Toronto, Ontario, Canada). In this report, the design criteria of the new scanner and its performance in noninvasive in vivo real-time mouse imaging are described. This scanner is unique in that it provides frequency selectivity over the range from 19 to 55 MHz, corresponding, respectively, to lateral resolutions ranging from 100 to 60 μm. For the first time, in this frequency range, the scanner combines imaging and Doppler blood flow sensing. Performance issues such as resolution and blood velocity sensitivity are described. Relevant applications in the mouse are given with images from day 5.5 of embryogenesis to adulthood. These images demonstrate the broad range of potential biologic applications of this novel imaging technology.

Section snippets

Instrument design

Figure 1a shows a schematic diagram of high-frequency US mouse imaging. The imaging system consists of a 3-D micropositioning scanhead that scans a high-frequency transducer over the field of view plus the associated signal- and imaging-processing hardware. The imaging process has been previously described in detail (Foster et al. 2000c). For mouse imaging, the 19- to 55-MHz center frequency transducer is moved linearly over the imaging field (8 mm × 8 mm), collecting US data at equally spaced

Experimental protocol for mouse imaging

All animal experimentation was performed under an approved animal care protocol. Timed pregnant CD-1 mice at various stages of development were lightly anesthetized with enflurane and imaged on a special mouse imaging stage that provided temperature feedback and heart rate monitoring (THM100 Indus Instruments, Houston, TX). After being anesthetized, the mouse abdomen was shaved and further cleaned with a chemical hair remover to minimize US attenuation. Imaging was performed while maintaining

Results

Examples of images made during early to mid mouse embryo development are given in Fig. 3. Figure 3a shows the earliest detection of the conceptus at day 5.5 of embryogenesis. At this stage, the inner cell mass (ICM), composed of the epiblast and primitive endoderm, and the trophectoderm have begun to form a cylindrical embryo. The embryo is visible as a diffuse bright region of approximately 250 μm in diameter in the lumen of the uterus. By day 7.5, the embryo has developed three distinct

Discussion and conclusion

US imaging has been scaled and optimized for the visualization of the living mouse. The resulting US biomicroscope has axial resolution on the order of 40 μm and lateral resolution ranging from 57 μm to 104 μm, depending on the choice of frequency. The current scanner (VisualSonics VS40) provides a field of view of 8 mm × 8 mm with a frame rate up to 10 Hz. The mouse UBM has the advantage of low cost, rapid imaging speed and portability. Real-time high-resolution imaging in combination with the

Acknowledgements

The authors acknowledge the financial support of the Canadian Institutes of Heath Research, the National Cancer Institute of Canada, the Terry Fox Foundation, and the Richard Ivey Foundation. Y. Q. Zhou acknowledges the personal support of the Ontario Research and Development Challenge Fund. The authors are grateful to Yong Lu for assistance with histology. F. S. Foster also wishes to acknowledge and disclose a financial interest in VisualSonics, the company now making this technology available

References (35)

  • M.D. Sherar et al.

    A 100 MHz B-scan ultrasound backscatter microscope

    Ultrason Imaging

    (1989)
  • D.H. Turnbull

    In utero ultrasound backscatter microscopy of early stage mouse embryos

    Comput Med Imaging Graphics

    (1999)
  • D.H. Turnbull et al.

    Ultrasound backscatter microscope analysis of mouse melanoma progression

    Ultrasound Med Biol

    (1996)
  • J. Battey et al.

    An action plan for mouse genomics and genetic resources

    Nature Genet

    (1999)
  • D.R. Bentley

    The human genome project—An overview

    Med Res Rev

    (2000)
  • S.K. Burley et al.

    Structural genomicsBeyond the human genome project

    Nature Genet

    (1999)
  • E.W. Cherin et al.

    Experimental characterization of fundamental and second harmonic beams for a high frequency ultrasound transducer

    Ultrasound Med Biol

    (2002)
  • Cited by (311)

    • Gastroprotective and gastric healing effects of the aqueous extract of Casearia sylvestris in rodents: Ultrasound, histological and biochemical analyzes

      2022, Journal of Ethnopharmacology
      Citation Excerpt :

      According to Heller et al. (2018), for an adequate evaluation of pharmacological effects, the anatomical, histological, and biochemical aspects and the body's physiological responses must be considered for the investigation of the test drug. Ultrasonography is a widely used method in clinical practice, and recent technological advances, such as the development of high-frequency probes, have allowed the realization of images in laboratory animals, including rodents (Foster et al., 2002). On examination, the gastric ulcer should be suspected in the presence of focal wall thickening, associated or not with mucosal irregularity or depression (Tomooka et al., 1989).

    View all citing articles on Scopus
    View full text