Modality Review: Introduction
to Ultrasound for Preclinical Imaging

The reflected portion of the transmitted soundwave depends heavily on the materials it interacts with, and that which it must pass through. Additionally, the angle at which the soundwave encounters the material will affect the reflection of the wave as well. This becomes important when considering the visualization of a specific structure in B-mode imaging. The strongest reflection of the soundwaves will occur when, for example, a blood vessel wall, is images directly perpendicular to the path of the soundwaves, to ensure the strongest reflection, and therefore best image. Additionally, soundwaves do not travel through air as efficiently as aqueous or water-based substances, such as ultrasound gel. Thus, ultrasound gel is important to act as an interface between the transducer and the imaging subject to ensure as much of the soundwave is transmitted to the imaging subject, and not absorbed into the air, which would weaken the resulting signal. 

Understanding the principles of soundwaves is important to understanding how ultrasound works, and an image is created. However, there are many different wavelengths, or frequencies, of sound that one could choose from. Lower frequency sound penetrates tissues and media well, and is not as susceptible to absorption in air, as higher frequencies, so at first glance would appear to be an ideal choice. However, the image resolution is directly correlated to the frequency of the soundwaves. Thus, for small animal imaging, where target organs or structures are quite small, then higher frequency soundwaves should be used. One must consider, however, the depth of penetration that is required to reach the target of interest and adjust the frequency of sound accordingly. 

When creating a 2-dimensional B-mode image, the system electronics receive the reflected soundwaves and consider the returning amplitude of the wave and interpret that into various levels of greyscale. The higher the amplitude of the returned wave, the brighter the greyscale value. In this way the system is able to look over many lines that make up the B-Mode image. When wishing to look along a single line, at a specific anatomical target (i.e., blood vessel wall, or myocardium) the user may locate the M-Mode sample volume in a specific location. Here the ultrasound system will display the movement of the tissue along that single line over time. In both B-Mode and M-Mode, the ultrasound system detects the amplitude of the reflected soundwave, to determine the greyscale intensity of the image, and looking at how that changes over time. 

Expanding B-mode capabilities from 2-dimenstions to 3, increased the utility of an ultrasound system to allow for volume calculations of many targets, for example specific organs, focal lesions within those organs including cysts, tumor, etc. This is accomplished on most preclinical imaging systems by attaching a programable motor to either the probe, or the animal handling platform. In either case, the user defines how far the motor should travel, and the gap between each acquired 2D B-mode image. The software then stacks the multiple 2D images together generating a 3D image. The user may then define the edges of the area of interest, and the system calculates the defined volume. 

Ultrasound imaging can also be used to assess blood flow, within a variety of blood vessels and within the chambers of the heart. The Doppler effect is key here – it states that frequency changes occur in the soundwaves if transmitters (i.e., piezoelectric crystals within the transducers) and receivers/reflectors (i.e., red blood cells within the blood vessels) move in relation to one another. A real-life example of this is the sound emitted by a firetruck as it moves towards a pedestrian; the frequency of the sound increases as the firetruck moves towards the pedestrian, while it decreases as the firetruck moves away from the pedestrian. When thinking about the Doppler effect with respect to blood flow, the ultrasound system transmits a known frequency of sound from the transducer, however the soundwave reflected off of a moving object, like a red blood cell, will be received at a different frequency. The ultrasound system must then determine the change in frequency of the soundwaves, thus providing an indication of both the velocity and direction that the blood cell was moving with. As one can imagine, the angle between the transmitted soundwave, and the movement of the red blood cell, for example, is of upmost importance to ensure the reflected soundwave returns to the transducer with the highest intensity. This angle becomes very important when working to quantify blood flow velocity over time, as is done with Pulsed Wave Doppler. 

The Doppler effect may be used to create a color flow map which is overlaid on a 2-dimensional B-mode image. Conventions exist, when displaying the color flow map in Color Doppler Mode, to display blood moving Away from the transducer in Blue, while blood moving Towards the transducer is Red. In the case where direction of blood flow is not relevant, for example in tumor imaging, Power Doppler may be used, in which no directional information is provided, the presence of blood is shown in shades of orange typically, with tones closer to white being of higher velocity then darker tones. If one wishes to examine the blood flow velocity within a specified sample volume, (i.e., within the inflow jet of blood flowing between the left atrium and the left ventricle, through the mitral valve, of the heart) then soundwaves only from this region are examined, and the resulting blood flow velocity is displayed over time, with directional information included. 

When discussing blood flow, as detected using the various Doppler techniques available, one is examining flow through large and smaller blood vessels, however this technique cannot be used to assess flow through the capillaries, for example. Flow in these small vessels, or perfusion within an organ or tumor, must be assessed using a contrast agent. Ultrasound contrast agents have traditionally been gas filled microbubbles, that is a bubble having a biocompatible outer shell (i.e., phospholipid) and a biocompatible gaseous core (i.e., perfluorocarbon gas). Due to their unique physical and acoustic characteristics the microbubbles can be imaged using ultrasound without destruction. When soundwaves of a low intensity/power strike a microbubble the rhythm of the acoustic wave causes the microbubbles to expand symmetrically through the negative and positive pressure waves created. In doing so, the microbubbles create an acoustic wave, of the same frequency of sound, which is received by the transducer, and can be converted into an image. When soundwaves or greater amplitude are used, the same phenomena occurs where the microbubbles expand and contract, however in this instance the microbubbles send back not only the same frequency of sound, but additional harmonic and subharmonic tones to that of the fundamental (or initial) frequency. Normal tissues on the other hand, do not create these harmonic and subharmonic tones. 

When considering microbubble imaging, there are two common approaches – harmonic imaging, and pulse inversion imaging. In harmonic imaging, filters are used to isolate the harmonic signal coming from the microbubbles, which is not emitted from the surrounding tissue. This, however, has some drawbacks including reduced spatial resolution and less contrast. However, if pulse inversion is used these drawbacks may be avoided. In pulse inversion, two sequential pulses are sent, one inverted with respect to the other. Tissue signals, which come back at the same frequency, will cancel each other out, while signal from microbubbles will not, resulting in a highly sensitive contrast agent imaging technique. 

One final technique, which some more advanced users are interested in is called shear wave elastography. In this technique, a secondary piezoelectric crystal is mounted onto the imaging probe, or specific crystals are programmed to create the additional soundwave. A non-imaging soundwave is sent into the tissue, and the reflected waves are received by the same crystal. The ultrasound system then determines the speed of sound through the specified 2-dimensional area of the tissue. A graphical representation of the calculated speeds is created, and the user may define specific regions of interest to quantify the speeds. The speed of sound through a specific tissue is dependent on the make-up of that tissue, down to the microscopic level. Elastography may be used to detect changes in tissue composition prior to changes in image characteristics that could be visualized. These tissue changes may be global changes, as may be seen in organ wide fibrosis, or accumulation of fat, or focal/regional changes.