Original contributionA new ultrasound instrument for in vivo microimaging of mice
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)
- et al.
40 MHz echocardiography scanner for cardiovascular assessment of mouse embryos
Ultrasound Med Biol
(1998) - et al.
High frequency pulsed Doppler ultrasound system for detecting and mapping blood flow in the microcirculation
Ultrasound Med Biol
(1997) Comparative genomicsThe key to understanding the human genome project
Bioessays
(1999)- et al.
Imaging, and spectrum analysis of contrast agents in the in vivo rabbit eye using very-high-frequency ultrasound
Ultrasound Med Biol
(1998) - et al.
Evaluation of ventricular and arterial hemodynamics in anesthetized closed-chest mice
J Am Soc Echocardiog
(1997) - et al.
Advances in ultrasound biomicroscopy
Ultrasound Med Biol
(2000) - et al.
High-frequency colour flow imaging of the microcirculation
Ultrasound Med Biol
(2000) - et al.
Alteration of limb and brain patterning in early mouse embryos by ultrasound-guided injection of Shh-expressing cells
Mech Dev
(1998) - et al.
Quantitative evaluation of left ventricular function in a transgenic mouse model of dilated cardiomyopathy with 2-dimensional contrast echocardiography
J Am Soc Echocardiog
(1999) - et al.
The design and fabrication of high frequency poly(vinylidene fluoride) transducers
Ultrason Imaging
(1989)
A 100 MHz B-scan ultrasound backscatter microscope
Ultrason Imaging
In utero ultrasound backscatter microscopy of early stage mouse embryos
Comput Med Imaging Graphics
Ultrasound backscatter microscope analysis of mouse melanoma progression
Ultrasound Med Biol
An action plan for mouse genomics and genetic resources
Nature Genet
The human genome project—An overview
Med Res Rev
Structural genomicsBeyond the human genome project
Nature Genet
Experimental characterization of fundamental and second harmonic beams for a high frequency ultrasound transducer
Ultrasound Med Biol
Cited by (311)
High-frequency ultrasound-guided intrathecal injections in a young mouse model: Targeting the central nervous system in drug delivery
2023, Journal of Neuroscience MethodsAnesthesia for in vivo imaging studies: practical considerations for experimental outcomes and animal welfare
2023, Anesthesia and Analgesia in Laboratory AnimalsGastroprotective and gastric healing effects of the aqueous extract of Casearia sylvestris in rodents: Ultrasound, histological and biochemical analyzes
2022, Journal of EthnopharmacologyCitation 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).
Externally triggered release of growth factors - A tissue regeneration approach
2021, Journal of Controlled ReleaseUltrahigh-Frequency Echocardiography of Autonomic Devoid Phox2B Homozygous Embryos Does Not Reveal a Significant Cardiac Phenotype before Embryo Death
2021, Ultrasound in Medicine and Biology